Modified cellulases with increased thermostability, thermophilicity, and alkalophilicity

ABSTRACT

A modified Family 6 cellulase enzyme comprising a proline residue at position 413 is provided. Genetic constructs and genetically modified microbes comprising DNA sequences encoding the modified Family 6 cellulase are also provided. Family 6 cellulases of the invention display improved thermostability, thermophilicity, alkalophilicity, or a combination thereof, relative to the parent Family 6 cellulases. Such cellulases find use in a variety of applications in industry that require cellulase stability and activities at temperatures, pH values, or both, above that of the native enzyme.

TECHNICAL FIELD

The present invention relates to modified cellulases. More specifically,the invention relates to modified Family 6 cellulases with improvedthermostability, alkalophilicity and/or thermophilicity. The presentinvention also relates to genetic constructs comprising nucleotidesequences encoding for modified Family 6 cellulases, methods for theproduction of the modified Family 6 cellulase from host strains and theuse of the modified Family 6 cellulases in the hydrolysis of cellulose.

BACKGROUND OF THE INVENTION

The most abundant polysaccharide in the biosphere, cellulose, consistsof D-glucose units linked together in linear chains via β-1,4 glycosidicbonds. These chains can vary in length and often consist of manythousands of units. Cellulose chains form numerous intra- andintermolecular hydrogen bonds, which result in the formation ofinsoluble cellulose microfibrils. This crystalline cellulose is arecalcitrant material with a natural half-life of over five millionyears.

In order to access this important renewable carbon source,microorganisms, such as bacteria and fungi, produce a cocktail ofenzymes to break down crystalline cellulose into glucose. Three generalclasses of cellulase enzymes act synergistically to hydrolyze thecrystalline cellulose into the simple energy source glucose.Endo-β-1,4-glucanases (EC 3.2.1.4) randomly hydrolyze amorphous regionsof crystalline cellulose generating oligosaccharides of various lengthsand consequently new chain ends. Cellobiohydrolases (orexo-β-1,4-cellobiohydrolase, EC 3.2.1.91) hydrolyze processivelycellobiose units from one end of the cellulose chain. Finally,β-1,4-glucosidases (EC 3.2.1.21) hydrolyse cellobiose into glucose.

Most cellobiohydrolases and endo-β-1,4-glucanases are multidomainproteins consisting of a catalytic core domain and a cellulose-bindingdomain separated by a flexible linker region. The cellulose-bindingdomain promotes adsorption of the enzyme to regions of the cellulosicsubstrate (Tomme, P., et al. 1988. Eur. J. Biochem 170:575-581; LehtioJ., et al. 2003 Proc. Natl. Acad. Sci. USA. 100:484-489), while thecatalytic core domain is responsible for the cleavage of cellulose. Thelinker region may ensure an optimal interdomain distance between thecore domain and the cellulose-binding domain (Srisodsuk M., et al. 1993.J. Biol. Chem. 268:20756-20761).

The catalytic domains are classified into the glycoside hydrolasefamilies based on amino acid sequence similarities whereby a familycomprises enzymes having similar fold and hydrolytic mechanisms but maydiffer in their substrate specificity. Trichoderma reesei contains knowncellulase genes for two cellobiohydrolases, i.e., Cel7A (also known asCBH1, which is a member of Family 7) and Cel6A (CBH2), at least eightendo-β-1,4-glucanases, i.e., Cel7B (EG1), Cel5A (EG2), Cel12A (EG3),Cel61A (EG4), Cel45A (EG5), Cel74A (EG6), Cel61B (EG7), and Cel5B (EG8),and at least seven β-1,4-glucosidase, i.e., Cel3A (BG1), CellA (BG2),Cel3B (BG3), Cel3C (BG4), CellB (BG5), Cel3D, and Cel3E (Foreman, P. K.,et al. 2003. J. Biol. Chem. 278:31988-31997).

T. reesei Cel6A (or TrCel6A) is one of the two major cellobiohydrolasessecreted by this fungus and has been shown to be efficient in theenzymatic hydrolysis of crystalline cellulose. TrCel6A is a member ofglycoside hydrolase Family 6, which comprises enzymes that hydrolyseβ-1,4 glycosidic bonds with inversion of anomeric configuration andincludes cellobiohydrolases as well as endo-β-1,4-glucanases. The threedimensional structures of TrCel6A (Rouvinen J., et al. 1990. Science249:380-386. Erratum in: Science 1990 249:1359), Thermobifida fuscaendo-β-1,4-glucanase Cel6A (TfCel6A, Spezio M., et al. 1993.Biochemistry. 32:9906-9916), Humicola insolens cellobiohydrolase Cel6A(HiCel6A, Varrot, A., et al. 1999 Biochem. J. 337:297-304), Humicolainsolens endo-β-1,4-glucanase Cel6B (HiCel6B, Davies, G. J., et al.2000. Biochem. J. 348:201-207), and Mycobacterium tuberculosis H37RvCel6A (MtCel6A, Varrot. A., et al. 2005. J. Biol. Chem. 280:20181-20184)are known.

Applications of cellulase enzymes in industrial processes are numerousand have proven commercially useful within the textile industry fordenim finishing and cotton softening; in the household and industrialdetergents for color brightening, softening, and soil removal; in thepulp and paper industries for smoothing fiber, enhancing drainage, andde-inking; in the food industry for extracting and clarifying juice fromfruits and vegetables, and for mashing; in the animal feed industry toimprove their nutritional quality; and also, in the conversion of plantfibers into glucose that are fermented and distilled to make low CO₂cellulose ethanol to reduce fossil fuel consumption, which is anemerging industry around the world (e.g. Gray K. A., et al. 2006. Curr.Opin. Chem. Biol. 10:141-146).

In order to obtain enzyme variants with improved stability properties,three strategies have generally been used within the art: 1) isolationof thermophilic enzymes from extremophiles, residing in severeenvironments such as extreme heat or cold, high salt concentrations orhigh or low pH conditions (e.g. U.S. Pat. No. 5,677,151 U.S. Pat. Appl.No. 20060053514); 2) protein engineering by rational design orsite-directed mutagenesis, which relies on sequence homology andstructural alignment within a family of proteins to identify potentiallybeneficial mutations using the principles of protein stability known inthe art (reviewed in: Eijsink, V. G., et al. 2004. J. Biotechnol.113:105-20.); and 3) directed evolution involving the construction of amutant library with selection or screening to identify improved variantsand involves a process of iterative cycles of producing variants withthe desired properties (recently reviewed in: Eijsink V G, et al. 2005.Biomol. Eng. 22:21-30). This approach requires no structural ormechanistic information and can uncover unexpected beneficial mutations.Combining the above strategies has proven to be an efficient way toidentify improved enzymes (Chica R. A., et al. 2005. Curr. Opin.Biotechnol. 16:378-384).

Using rational design, Zhang et al. (Zhan S et al., 2000. Eur. J.Biochem. 267:3101-15), introduced a new disulfide bond across the N- andC-terminal loops from TfCel6B using two double mutations, and fourglycine residue mutations were chosen to improve thermostability. Noneof the mutations increased thermostability of this cellobiohydrolase andmost mutations reduced thermostability by 5-10° C. Surprisingly, thedouble mutation N233C-D506C showed a decrease of 10° C. for the T₅₀(Zhang S et al., 2000. Eur. J. Biochem. 267:3101-15), or a slightincrease of about 2° C. for the T₅₀ (Ai, Y. C. and Wilson, D. B. 2002.Enzyme Microb. Technol. 30:804-808). Wohlfahrt (Wohlfahrt, G., et al.2003. Biochemistry. 42:10095-10103) disclosed an increase in thethermostability of TrCel6A, at an alkaline pH range, by replacingcarboxyl-carboxylate pairs into amide-carboxylate pairs. A singlemutant, E107Q, and a triple mutant, E107Q/D170N/D366N, have an improvedT_(m) above pH 7 but a lower T_(m) at pH 5, which is the optimal pH ofthe wild-type TrCel6A. These mutations are found in, or close to, the N-and C-terminal loops. Hughes et al (Hughes, S. R., et al. 2006. ProteomeSci. 4:10-23) disclose a directed evolution strategy to screenmutagenized clones of the Orpinomyces PC-2 cellulase F (OPC2Cel6F) withtargeted variations in the last four codons for improved activity atlower pH, and identified two mutants having improved activity at lowerpH and improved thermostability.

The above reports describing rational design of Family 6 cellulasessuggest that the introduction of hydrogen or disulfide bonds into theC-terminal loops is not a good strategy to increase the thermostabilityat optimal hydrolysis conditions. Furthermore, stabilizing the exo-loopof the T. reesei Family 7 cellobiohydrolase Cel7A, which forms the roofof the active site tunnel, by introducing a disulfide bond with mutationD241C/D249C showed no improvement in thermostability (von Ossowski I.,et al. 2003. J. Mol. Biol. 333:817-829).

TrCel6A variants with improved thermostability are described in USPatent Publication No. 20060205042. Mutations were identified basedalignment of TrCel6A amino acid sequence with those of eight Family 6members using structural information and a modeling program. Thisalignment served as basis for the determination of a so-called consensussequence. Those mutations that, according to the 3D-structure model ofTrCel6A, fit into the structure without disturbance and were likely toimprove the thermostability of the enzyme were selected as replacementfor improved thermostability of TrCel6A. Among those identified asimproving the thermostability of TrCel6A was the mutation of the serineat position 413 to a tyrosin (S413Y). This mutation increased theretention of enzymatic activity after a 1 hour pre-incubation at 61° C.from 20-23% for the parental TrCel6A to 39-43% for TrCel6A-S413Y;however, after a 1 hour pre-incubation at 65° C., the parent TrCel6Aretained 5-9% of its activity while TrCel6A-S413Y retained 6-8% of itsactivity. The melting temperature, or Tm, improved by 0.2-0.3° C., from66.5° C. for the parental TrCel6A to 66.7-66.8° C. for TrCel6A-S413Y.

Despite knowledge of the mechanisms of and desirable attributes forcellulases in the above and related industrial applications, thedevelopment of thermostable cellulases with improved stability,catalytic properties, or both improved stability and catalyticproperties, would be advantageous. Although thermophilic andthermostable enzymes may be found in nature, the difficulty in achievingcost-effective large-scale production of these enzymes has limited theirpenetration into markets for industrial use. Therefore, a need existsfor improved stable cellulases which can be economically produced at ahigh-level of expression by industrial micro-organisms such as T.reesei.

SUMMARY OF THE INVENTION

The present invention relates to modified Family 6 cellulases. Morespecifically, the invention relates to modified Family 6 cellulases thatexhibit enhanced thermostability, alkalophilicity and/orthermophilicity. The present invention also relates to geneticconstructs comprising nucleotide sequences encoding for modified Family6 cellulases, methods for the production of the modified Family 6cellulase from host strains and the use of the modified Family 6cellulases in the hydrolysis of cellulose.

It is an object of the invention to provide an improved cellulase withincreased thermostability, thermophilicity and alklophilicity.

This invention relates to a modified Family 6 cellulase produced bysubstitution of an amino acid at position 413 with a proline. Theposition(s) of the amino acid substitution(s) are determined fromsequence alignment of the modified cellulase with a Trichoderma reeseiCel6A amino acid sequence as defined in SEQ ID NO: 1. The modifiedFamily 6 cellulase exhibits enhanced thermostability, alkalophilicity,thermophilicity, or a combination thereof, relative to a parent Family 6cellulase from which the Family 6 cellulase is derived.

The modified Family 6 cellulase may be derived from a filamentousfungus, such as Trichoderma reesei. In one embodiment of the invention,the modified cellulase is not derived from a cellulase which has anaturally-occurring proline residue at position 413 (TrCel6A numbering),for example a native Family 6 cellulase (CelF from Orpinomyces sp PC-2)which contains a proline residue at position 413.

This invention also includes a modified Family 6 cellulase comprising aproline residue at position 413 and further comprising polar amino acidsat positions selected from 231, 305, 410 or a combination thereof.

The present invention also pertains to the modified Family 6 cellulasecomprising a proline at position 413 and further comprising asubstituted amino acid at position 231 selected from the groupconsisting of Ser, or Thr. The substituted amino acid at position 231may be Ser.

The present invention also pertains to the modified Family 6 cellulasecomprising a proline at position 413 and further comprising asubstituted amino acid at position 305 selected from the groupconsisting of Ser and Thr.

The present invention also pertains to the modified Family 6 cellulasecomprising a proline residue at position 413 and further comprising asubstituted amino acid at position 410 selected from the groupconsisting of Gln and Asn.

The present invention also includes a Family 6 cellulase comprising aproline residue at position 413 and further comprising substituted aminoacids at positions 231 and 305 with Ser residues (i.e. 231S, 305S), andsubstitution of an amino acid at position 410 with Gln. The modifiedFamily 6 cellulase comprising these mutations may be from a filamentousfungus, such as Trichoderma reesei.

The present invention also relates to a modified Family 6 cellulasecomprising a proline residue a position 413 and having an increase inthermostability relative to a parent cellulase, as measured by the“T₅₀”, from about 5° C. to about 30° C. higher, or from about 9° C. toabout 20° C. higher than the corresponding parent cellulase.

The present invention also relates to a modified Family 6 cellulasecomprising a proline residue at position 413 and having an increase inits temperature for maximal activity (T_(opt)) of from about 1.5° C. toabout 30° C. higher, or from about or 2.5° C. to about 20° C. higher,that the T_(opt) of a parent Family 6 celulase. The present inventionalso relates to a modified Family 6 cellulase comprising a prolineresidue at position 413 and having an increase in its pH for maximalactivity (pH_(opt)) of about 0.5 units to about 6.0 units higher,relative to a parent cellulase.

The present invention also relates to a modified Family 6 cellulaseselected from the group consisting of:

(SEQ ID NO: 12) TrCe16A-S413P; (SEQ ID NO: 13)TrCe16A-G82E-G231S-N305S-R410Q-S413P; (SEQ ID NO: 14)TrCe16A-G231S-S413P; (SEQ ID NO: 15) TrCe16A-N305S-S413P; (SEQ ID NO:16) TrCe16A-R410Q-S413P; (SEQ ID NO: 17) TrCe16A-G231S-N305S-S413P; (SEQID NO: 18) TrCe16A-G231S-R410Q-S413P; (SEQ ID NO: 19)TrCe16A-N305S-R410Q-S413P; (SEQ ID NO: 20)TrCe16A-G231S-N305S-R410Q-S413P; (SEQ ID NO: 21) HiCe16A-Y420P; and (SEQID NO: 22) PcCe16A-S407P.

The invention also relates to genetic constructs for directingexpression and secretion of the modified Family 6 cellulase from a hostmicrobe including, but not limited to, strains of Trichoderma reesei.

The present invention relates to a genetic construct comprising a DNAsequence encoding a modified Family 6 cellulase comprising a prolineresidue at position 413, which DNA sequence is operably linked to DNAsequences regulating its expression and secretion from a host microbe.Preferably, the DNA sequences regulating the expression and secretion ofthe modified Family 6 cellulase are derived from the host microbe usedfor expression of the modified cellulase. The host microbe may be ayeast, such as Saccharomyces cerevisiae, or a filamentous fungus, suchas Trichoderma reesei.

The invention also relates to a genetic construct comprising a DNAsequence encoding a modified Family 6 cellulase comprising a prolineresidue at position 413 and further comprising substituted amino acidsat positions 231 and 305 with Ser and substitution of an amino acid atposition 410 with Gln. The DNA sequence is operably linked to DNAsequences regulating its expression and secretion from a host microbe.Preferably, the DNA sequences regulating the expression and secretion ofthe modified Family 6 cellulase are derived from a filamentous fungus,including, but not limited to, Trichoderma reesei.

The invention also relates to a genetically modified microbe capable ofexpression and secretion of a modified Family 6 cellulase comprising aproline residue at position 413 and comprising a genetic constructencoding the modified Family 6 cellulase. In one embodiment, themodified Family 6 cellulase further comprises Ser residues at positions231 and 305 and a Gln residue at position 410. Preferably, thegenetically modified microbe is a yeast or filamentous fungus. Thegenetically modified microbe may be a species of Saccharomyces, Pichia,Hansenula, Trichoderma, Aspergillus, Fusarium, Humicola, Neurospora orPhanerochaete.

The present invention also relates to the use of a modified Family 6cellulase comprising a proline residue at position 413 for treatment ofa cellulosic substrate.

The invention also relates to the process of producing the modifiedFamily 6 cellulase, including transformation of a yeast or fungal host,selection of recombinant yeast or fungal strains expressing the modifiedFamily 6 cellulase, and culturing the selected recombinant strains insubmerged liquid fermentations under conditions that induce theexpression of the modified Family 6 cellulase.

Family 6 cellulases of the present invention comprising a prolineresidue at position 413 display improved thermostability andthermophilicity or alkalophilicity relative to wild-type Family 6cellulases. Without wishing to be bound by theory, improvedthermostability of the modified Family 6 cellulase results from aminoacid substitutions that stabilize the C-terminal loop of Family 6cellobiohydrolases by increasing the stability of the small α-helix.

Such cellulases find use in a variety of applications in industry thatrequire enzyme stability and activities at temperatures and/or pH valuesabove that of the native enzyme. For example, modified Family 6cellulases, as described herein, may be used for the purposes ofsaccharification of lignocellulosic feedstocks for the production offermentable sugars and fuel alcohol, improving the digestibility offeeds in ruminant and non-ruminant animals, pulp and paper processing,releasing dye from and softening denim.

This summary of the invention does not necessarily describe all featuresof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows an amino acid sequence alignment among Family 6 cellulases.The amino acid numbering for each cellulase is compared with that of theTrichoderma reesei Cel6A (TrCel6A; SEQ ID NO:1) as indicated at the leftand right of each sequences. The residues at positions 213, 305, 410 and413 (relative to TrCel6A) are indicated with an asterisk. The residuesidentical with the corresponding amino acid in TrCel6A are in bold. Forcellulases with a cellulose-binding domain, only the catalytic coresequences are presented. CfCel6B (SEQ ID NO:2); HiCel6A (SEQ ID NO:4);HiCel6B (SEQ ID NO:11); MtCel6A (SEQ ID NO:9); NpCel6A (SEQ ID NO:5);OpC2Cel6F (SEQ ID NO:6); PE2Cel6A (SEQ ID NO:8); TfCel6A (SEQ ID NO:10);TfCel6B (SEQ ID NO:3).

FIG. 2 depicts plasmid vectors a) YEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2vector, b) YEpFLAGΔKpn10-cbh2 directing the expression and secretion ofnative and modified TrCel6A from recombinant Saccharomyces cerevisiae(The same organization if found for the TrCel6 variants cloned in thesame vectors), c) YEpFLAGΔKpn10-PcCel6A directing the expression andsecretion of native and modified PcCel6A from recombinant Saccharomycescerevisiae (The same organization if found for the PcCel6 variantscloned in the same vectors), d) YEpFLAGΔKpn10-HiCel6A directing theexpression and secretion of native and modified HiCel6A from recombinantSaccharomyces cerevisiae (The same organization if found for the HiCel6variants cloned in the same vectors).

FIG. 3 depicts the vector pC/X—S413P-TV used to transform and direct theexpression and secretion of modified TrCel6A from recombinantTrichoderma reesei. As shown, the TrCel6A-S413P gene is operable linkedto the promoter of the cbh1 (TrCel7A) gene, the secretion signal peptideof the xln2 (TrXyl11B) genes and the transcriptional terminator of thenative cbh2 (TrCel6A) gene. The selection marker is the Neurosporacrassa pyr4 gene.

FIG. 4 shows the effect of pre-incubation temperature on the relativeresidual activity (%), as measured by the release of reducing sugarsfrom β-glucan in a 30 minutes assay at a) 65° C., of the native TrCel6Aand modified Family 6 cellulases TrCel6A-S413P, TrCel6A-R410Q-S413P,TrCel6A-G231 S-N305S-S413P, TrCel6A-G231S-R410Q-S413P,TrCel6A-N305S-R410Q-S413P and TrCel6A-G231S-N305S-R410Q-S413P, b) 60°C., of the native PcCel6A and modified Family 6 cellulasesPcCel6A-S407P, c) 65° C., of the native HiCel6A and modified Family 6cellulases HiCel6A-Y420P, after 15 minutes incubation at temperaturesbetween 45° C. and 75° C.

FIG. 5 shows the effect of increasing pre-incubation times on therelative residual activity (%), as measured by the release of reducingsugars a soluble β-glucan substrate in a 30 minutes assay at a) 65° C.,of the native TrCel6A and modified Family 6 cellulases TrCel6A-S413P,TrCel6A-R410Q-S413P, TrCel6A-G231S-N305S-S413P,TrCel6A-G231S-R410Q-S413P, TrCel6A-N305S-R410Q-S413P andTrCel6A-G2311S-N305S-R410Q-S413P after 0-120 minutes incubation at 60°C. and b) 60° C., of the native PcCel6A and modified Family 6 cellulasesPcCel6A-S407P after 0-120 minutes incubation at 55° C.

FIG. 6 shows the effect of temperature on the enzymatic activity of a)the native TrCel6A and modified Family 6 cellulases TrCel6A-S413P,TrCel6A-G82E-G231S-N305S-R410Q-S413P, TrCel6A-R410Q-S413P,TrCel6A-G231S-N305S-S413P, TrCel6A-G231S-R410Q-S413P,TrCel6A-N305S-R410Q-S413P and TrCel6A-G231S-N305S-R410Q-S413P b) thenative PcCel6A and modified Family 6 cellulases PcCel6A-S407P and c) thenative HiCel6A and modified Family 6 cellulases HiCel6A-Y420P during 30minutes incubation at pH 5.0. The data are normalized to the activityobserved at the temperature optimum for each enzyme.

FIG. 7 shows the effect of pH on the enzymatic activity of a) the nativeTrCel6A and modified Family 6 cellulases TrCel6A-S413P,TrCel6A-G82E-G231S-N305S-R410Q-S413P, TrCel6A-R410Q-S413P,TrCel6A-G231S-N305S-S413P, TrCel6A-G231S-R410Q-S413P,TrCel6A-N305S-R410Q-S413P and TrCel6A-G231S-N305S-R410Q-S413P b) thenative PcCel6A and modified Family 6 cellulases PcCel6A-S407P and c) thenative HiCel6A and modified Family 6 cellulases HiCel6A-Y420P during 30minutes incubation at pH 3.95-7.45. The data are normalized to theactivity observed at the pH optimum for each enzyme.

FIG. 8 shows the relative activity of whole Trichoderma cellulasescomprising TrCel6A or TrCel6A-S413P (along with all of the remainingnative Trichoderma reesei cellulase components) in the enzymatichydrolysis of pretreated lignocellulosic substrate after 0, 4, 20.5, 28,40.5, 52, 68, 76 and 96 hours of pre-incubation in the absence ofsubstrate at 50° C. in 50 mM citrate buffer, pH 5.0.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to modified cellulase. More specifically,the invention relates to modified Family 6 cellulases with enhancedthermostability, alkalophilicity and/or thermophilicity. The presentinvention also relates to genetic constructs comprising nucleotidesequences encoding for modified Family 6 cellulases, methods for theproduction of the modified Family 6 cellulase from host strains and theuse of the modified Family 6 cellulases in the hydrolysis of cellulose.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

Modified Family 6 Cellulases

Family 6 (previously, Family B) cellulases enzymes are a group ofenzymes that hydrolyse the β-1,4 glucosidic linkages in cellulose withinversion of configuration of the anomeric carbon (Claeyssens, M. andHenrissat, B. 1992, Protein Science 1: 1293-1297). Family 6 cellulasesshare extensive amino acid sequence similarity (FIG. 1). A cellulase isclassified as a Family 6 cellulase if it comprises amino acids common toother Family 6 cellulase, including two aspartic acid (D) residues whichmay serve as catalytic residues. These aspartic acid residues are foundat positions 175 and 221 (see FIG. 1; based on TrCel6A (Trichodermareesei Cel6A enzyme) amino acid numbering). Most of the Family 6cellulases identified thus far are mesophilic. However, this family alsoincludes thermostable cellulases from Thermobifida fusca (TfCel6A andTfCel6B) and the alkalophilic cellulases from Humicola insolens (HiCel6Aand HiCel6B).

The topology of Family 6 catalytic domains is a variant of theα/β-barrel with a central β-barrel containing seven parallel β-strandsconnected by five α-helices. One important difference between Family 6cellobiohydrolases and endo-β-1,4-glucanases is the length of their N-and C-terminal loops present on each side of the active site and whichare responsible for their functional behavior on cellulose. In thecellobiohydrolases, an extensive C-terminal loop forms a tunnel with theN-terminal loop enclosing the active site. This confers the uniqueproperty of cellobiohydrolases to attack the ends of crystallinecellulose where the N- and C-terminal loops maintain a single cellulosechain in the active site and facilitate the processive degradation ofthe substrate. In the endo-β-1,4-glucanases, the C-terminal loop isreduced in length and the N-terminal loop pulls it away from the activesite and could be also shorter resulting in a more open active siteallowing access to internal β-1,4 glycosidic bonds of cellulose forhydrolysis. The role of these loops in the functional behavior of Family6 enzymes on cellulose was confirmed by the deletion of fifteen aminoacids of the C-terminal loop of the Cellulomonas fimi cellobiohydrolaseCel6B in order to mimic the properties of an endo-β-1,4-glucanase(Meinke A., et al. 1995. J. Biol. Chem. 270:4383-4386). The mutationenhanced the endo-β-1,4-glucanase activity of the enzyme on solublecellulose, such as carboxymethylcellulose, and altered itscellobiohydrolase activity on insoluble cellulose.

Non-limiting examples of Family 6 cellulases that may be modifiedfollowing the general approach and methodology as outlined herein aredescribed in Table 1 below.

TABLE 1 Family 6 cellulase enzymes Microbe Cellulase SEQ ID No.Cellulomonia fimi CfCe16B 2 Humicola insolens HiCe16A 4 Humicolainsolens HiCe16B 11  Mycobacteriumn tuberculosis MtCe16A 9Neocallimatrix patriciarum NpCe16A 5 Orpinomyces sp. PC-2 OpC2Ce16F 6Phanerochaete chrysosporium PcCe16A 7 Pyromyces sp. E2 PE2Ce16A 8Thermobifida fusca TfCe16A 10  Thermobifida fusca TfCe16B 3

Examples of preferred Family 6 cellulases, which are not meant to belimiting, include Trichoderma reesei Cel6A, Humicola insolens Cel6A,Phanerochaete chrysosporium Cel6A, Cellulomonas fimi Cel6B, Thermobifidafusca Cel6B. More preferably, the modified cellulase of the presentinvention comprises a modified Trichoderma reesei Cel6A enzyme.

By “modified Family 6 cellulase” or “modified cellulase”, it is meant aFamily 6 cellulase in which the amino acid at position 413 (saidposition determined from sequence alignment of said modified cellulasewith a Trichoderma reesei Cel6A amino acid sequence as defined in SEQ IDNO:1) has been altered, using techniques that are known to one of skillin the art, to a proline and which exhibits improvements inthermostability, thermophilicity, alkalophilicity, or a combinationthereof, over the corresponding unmodified Family 6 cellulase.Techniques for altering amino acid sequences include, but are notlimited to, site-directed mutagenesis, cassette mutagenesis, randommutagenesis, synthetic oligonucleotide construction, cloning and othergenetic engineering techniques (Eijsink V G, et al. 2005. Biomol. Eng.22:21-30, which is incorporated here in by reference). It will beunderstood that the modified cellulase may be derived from any Family 6cellulase. The modified cellulase may be derived from a wild-typecellulase or from a cellulase that already contains other amino acidsubstitutions.

For the purposes of the present invention, the parent cellulase is acellulase that does not contain a substitution of its original aminoacid at position 413 (said position determined from sequence alignmentof said modified cellulase with a Trichoderma reesei Cel6A amino acidsequence as defined in SEQ ID NO:1) by a proline and is otherwiseidentical to the modified cellulase. As such, the parent cellulase maybe a cellulase that contains amino acid substitutions at other positionsthat have been introduced by genetic engineering or other techniques.However, a parent cellulase does not include those cellulases in whichthe naturally occurring amino acid at position 413 is a proline.

By “TrCel6A numbering”, it is meant the numbering corresponding to theposition of amino acids based on the amino acid sequence of TrCel6A(Table 1; FIG. 1; SEQ ID NO:1). As disclosed below, and as is evident byFIG. 1, Family 6 cellulases exhibit a substantial degree of sequencesimilarity. Therefore, by aligning the amino acids to optimize thesequence similarity between cellulase enzymes, and by using the aminoacid numbering of TrCel6A as the basis for numbering, the positions ofamino acids within other cellulase enzymes can be determined relative toTrCel6A.

Enzyme thermostability can be defined by its melting temperature(T_(m)), the half-life (t_(1/2)) at defined temperature, and thetemperature at which 50% of the initial enzyme activity is lost afterincubation at defined time (T₅₀). Thermophilic enzymes typically showcommon structural elements that have been identified as contributingfactors to enzyme thermostability when compared to their mesophiliccounterparts (e.g. see Sadeghi M., et al. 2006. Biophys. Chem.119:256-270). These structural elements include greater hydrophobicity,better packing, increased polar surface area, deletion or shortening ofloops, interactions, smaller and less numerous cavities, stability ofα-helix, increase in aromatic interactions, additional disulfide bridgesor metal binding and glycosylation sites, decreased glycines andenhanced prolines content, increased hydrogen bonding and salt bridges,improved electrostatic interactions, decreased of thermolabile residues,and conformational strain release.

For the purposes of the present invention, a cellulase exhibits improvedthermostability with respect to a corresponding parent cellulase if ithas a T₅₀ which is at least about 4° C., or at least about 9° C. higherthan that of the parent cellulase, or for example a cellulase having aT₅₀ from about 4° C. to about 30° C. higher, or any amount therebetween,or a T₅₀ from about 9° C. to about 30° C. higher, or any amounttherebetween, when compared to that of the parent cellulase. The T₅₀ isthe temperature at which the modified or the natural enzyme retains 50%of its residual activity after a pre-incubation for 15 minutes and isdetermined by the assay detailed in Example 10.4. As set forth inExample 10.4, the residual activity against β-glucan in a 30 minuteassay at 65° C. is normalized to 100%.

The modified Family 6 cellulase may have Tso which is about 4° C. toabout 30° C. higher than that of a corresponding parent cellulase, orany range therebetween, about 5° C. to about 20° C. higher, or any rangetherebetween, about 8° C. to about 15° C. higher, or any rangetherebetween, or from about 9° C. to about 15° C. higher, or any rangetherebetween. For example, the modified cellulase may have a T₅₀ that isat least about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,or 30° C. higher than that of the corresponding parent cellulase.

The modified Family 6 cellulase may also be characterized as having aT₅₀ above 65° C. (or at least 5° C. above that of the correspondingparent Family 6 cellulase), for example, the modified cellulase may havea T₅₀ from about 65° C. to about 90° C., or any amount therebetween. Themodified Family 6 cellulase may have a T₅₀ above 70° C. (or at least 9°C. above the parent Family 6 cellulase) for example, the modifiedcellulase may have a T₅₀ so from about 70° C. to about 90° C., or anyamount therebetween. The Family 6 cellulase may have a T₅₀ of 50, 55,60, 65, 70, 75, 80, 85 or 90° C. or any amount therebetween.

For the purposes of this specification, a cellulase exhibits improvedthermophilicity with respect to a corresponding parent cellulase if thecellulase exhibits a temperature optimum (T_(opt)) that is at leastabout 1.5° C. higher than the T_(opt) of the corresponding parentcellulase. For example, a cellulase exhibits improved thermophilicity ifthe cellulase exhibits a temperature optimum (T_(opt)) that is fromabout 1.5° C. to about 30° C. or any amount therebetween, higher thanthe T_(opt) of the corresponding parent cellulase By temperature optimumor T_(opt), it is meant the highest temperature at which a cellulaseexhibits its maximal activity. For the purposes of this specification,the T_(opt) of a Family 6 cellulase is determined by measuring thetemperature profile of activity against a β-glucan substrate as detailedin Example 10.1. The temperature profile for the activity of thecellulase is measured at its pH optimum.

The modified Family 6 cellulase may have a T_(opt) which is at leastabout 1.5° C. to about 30° C. higher than the T_(opt) of a correspondingparent Family 6 cellulase. In a preferred embodiment, the T_(opt) of themodified Family 6 cellulase is at least about 2.5° C. to about 20° C.higher than the T_(opt) of parent Family 6 cellulase. For example, themodified Family 6 cellulase may have a T_(opt) of at least about 1.5,2.5, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 15.0, 20.0, 25.0, or 30° C. higherthan that of the corresponding parent cellulase.

The terms “thermostability” and “thermophilicity” have been usedinterchangeably within the literature. However, the use of the terms asdefined herein is consistent with the usage of the terms in the art(Mathrani, I and Ahring, B. K. 1992 Appl. Microbiol. Biotechnol.38:23-27).

For the purposes of the present invention, a cellulase exhibits improvedalkalophilicity with respect to a corresponding parent cellulase if thecellulase exhibits a pH_(opt) that is at least about 0.5 units higherthan the pH_(opt) of the parent cellulase. By pH_(opt), it is meant thehighest pH at which a cellulase exhibits its maximal activity. For thepurpose of this specification, the pH_(opt) is determined by measuringthe pH profile of a Family 6 cellulase as set out in Example 10.2.

The modified Family 6 cellulase may have a pH_(opt) that is at leastabout 0.5 units to about 6.0 units, or any amount therebetween, higherthan the pH_(opt) of the parent Family 6 cellulase. In a preferredembodiment, the pH_(opt), of the modified Family 6 cellulase is at leastabout 0.8 units to about 5.0 units, or any amount therebetween, higherthan the pH_(opt) parent Family 6 cellulase. For example, the pH_(opt)of the cellulase may be about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5 or 6.0 units higher than the pH_(opt) of the parentcellulase.

As described in more detail herein, several mutant Family 6 cellulaseshave been prepared that exhibit enhanced thermostability,thermophilicity, alkalophilicity, or a combination thereof. A list ofseveral mutants, which is not to be considered limiting in any manner,is presented in Table 2.

TABLE 2 Modified Family 6 cellulases New mutant TrCe16A SEQ ID NO:TrCe16A-S413P 12 TrCe16A-G82E-G231S-N305S-R410Q-S413P 13TrCe16A-G231S-S413P 14 TrCe16A-N305S-S413P 15 TrCe16A-R410Q-S413P 16TrCe16A-G231S-N305S-S413P 17 TrCe16A-G231S-R410Q-S413P 18TrCe16A-N305S-R410Q-S413P 19 TrCe16A-G231S-N305S-R410Q-S413P 20HiCe16A-Y420P 21 PcCe16A-S407P 22Genetic Constructs Comprising Modified Family 6 cellulases

The present invention also relates to genetic constructs comprising aDNA sequence encoding the modified Family 6 cellulase operably linked toregulatory DNA sequences directing the expression and secretion of themodified Family 6 cellulase from a host microbe. The regulatorysequences are preferably functional in a fungal host. The regulatorysequences may be derived from genes that are highly expressed andsecreted in the host microbe under industrial fermentation conditions.In a preferred embodiment, the regulatory sequences are derived from anyone or more of the Trichoderma reesei cellulase or hemicellulase genes.

The genetic construct may further comprise a selectable marker to enableisolation of a genetically modified microbe transformed with theconstruct as is commonly known with the art. The selectable marker mayconfer resistance to an antibiotic or the ability to grow on mediumlacking a specific nutrient to the host organism that otherwise couldnot grow under these conditions. The present invention is not limited bythe choice of selection marker, and one of skill may readily determinean appropriate marker. In a preferred embodiment, the selection markerconfers resistance to hygromycin, phleomycin, kanamycin, geneticin, orG418, complements a deficiency of the host microbe in one of the trp,arg, leu, pyr4, pyr2, ura3, ura5, his, or ade genes or confers theability to grow on acetamide as a sole nitrogen source. In a morepreferred embodiment, the selectable marker is the Neurospora crassapyr4 gene encoding orotidine-5′-decarboxylase.

Genetically Modified Microbes Comprising Modified Family 6 cellulases

The modified Family 6 cellulase may be expressed and secreted from agenetically modified microbe produced by transformation of a hostmicrobe with a genetic construct encoding the modified Family 6cellulase. The host microbe is preferably a yeast or a filamentousfungi, including, but not limited to, a species of Saccharomyces,Pichia, Hansenula, Trichoderma, Hypocrea, Aspergillus, Fusarium,Humicola, Neurospora or Phanerochaete. Typically, the host microbe isone from which the gene(s) encoding any or all Family 6 cellulases havebeen deleted. In a most preferred embodiment, the host microbe is anindustrial strain of Trichoderma reesei.

The genetic construct may be introduced into the host microbe by anynumber of methods known by one skilled in the art of microbialtransformation, including but not limited to, treatment of cells withCaCl₂, electroporation, biolistic bombardment, PEG-mediated fusion ofprotoplasts (e.g. White et al., WO 2005/093072, which is incorporatedherein by reference).

After selecting the recombinant fungal strains expressing the modifiedFamily 6 cellulase, the selected recombinant strains may be cultured insubmerged liquid fermentations under conditions that induce theexpression of the modified Family 6 cellulase.

Hydrolysis of Cellulosic Substrates

The present invention also relates to the use of the modified Family 6cellulases described herein for the hydrolysis of a cellulosicsubstrate. By the term “cellulosic substrate”, it is meant any substratederived from plant biomass and comprising cellulose, including, but notlimited to, lignocellulosic feedstocks for the production of ethanol orother high value products, animal feeds, forestry waste products, suchas pulp and wood chips, and textiles.

By the term “lignocellulosic feedstock”, it is meant any type of plantbiomass such as, but not limited to, non-woody plant biomass, cultivatedcrops such as, but not limited to, grasses, for example, but not limitedto, C4 grasses, such as switch grass, cord grass, rye grass, miscanthus,reed canary grass, or a combination thereof, sugar processing residues,for example, but not limited to, baggase, beet pulp, or a combinationthereof, agricultural residues, for example, but not limited to, soybeanstover, corn stover, rice straw, rice hulls, barley straw, corn cobs,wheat straw, canola straw, oat straw, oat hulls, corn fiber, or acombination thereof, forestry biomass for example, but not limited to,recycled wood pulp fiber, sawdust, hardwood, for example aspen wood,softwood, or a combination thereof.

In the saccharification of lignocellulosic feedstocks for the productionof ethanol, or other products, cellulases of the invention may be usedto hydrolyze a pretreated feedstock produced by, for example, but notlimited to, steam explosion (see Foody, U.S. Pat. No. 4,461,648, whichis incorporated herein by reference and to which the reader is directedfor reference). Pretreatment may involve treatment of the feedstock withsteam, acid, or typically a combination of steam and acid, such that thecellulose surface area is greatly increased as the fibrous feedstock isconverted to a muddy texture, with little conversion of the cellulose toglucose. The cellulase enzymes of the invention then may be used tohydrolyze cellulose to glucose in a subsequent step. The glucose maythen be converted to ethanol or other products.

Modified cellulase enzymes of the invention may be added to pulp or woodchips to enhance the bleaching or reduce refining energy of the pulp.The pulp may be produced by a chemical pulping process or by mechanicalrefining.

Increasing the Thermostability of Family 6 Cellulases

The thermostability of the mutant Family 6 cellulase was compared viapre-incubation of the enzyme in the absence of substrate at differenttemperatures. After 15 minutes, the residual activity of the cellulasewas determined via a standard assay with soluble β-glucan as asubstrate.

The effect of the S413P mutation, alone or in combination with one ormore of G231S, N305S and R410Q, on the thermostability of Family 6cellulase was determined via a comparative study of the modifiedTrCel6A-S413P and the parent TrCel6A. After pre-incubation at highertemperatures for up to 120 minutes, the former retained greater residualactivity than the latter (FIG. 5 a).

The pre-incubation temperature that allowed Family 6 cellulase to retain50% of the residual activity, T₅₀, was determined. For the modifiedFamily 6 cellulase, TrCel6A-S413P, the T₅₀ was 64.1° C., as compared to59° C. for the parent TrCel6A (FIG. 4 a). This represented an increasein the thermostability by over 5° C. through the introduction of theS413P mutation.

The T₅₀ of the other TrCel6A variants was at least 3.2° C. higher thenwild-type TrCel6A. PcCel6A-S407P and Hicel6A-Y420P also have shown anincrease in T₅₀ when compared to their respective parent enzyme (FIGS. 4b and c).

Increasing the Thermophilicity of Family 6 Cellulases

The thermophilicity of the modified Family 6 cellulases was determinedby measuring effect of the assay temperature on the hydrolysis ofβ-glucan.

All modified Family 6 cellulases shown an improved T_(opt) for β-glucanhydrolysis when compared to their respective wild-type except variantTrCel6A-G231S-N305S-R410Q-S413P which on the other hand exhibits a broadtemperature range with more then 80% of the maximum activity (FIG. 6).Among all TrCel6A variants, TrCel6A-S413P has the higher optimaltemperature at 72.2° C., an increase of 5.6° C. in thermophilicitycompared to wild-type TrCel6A (FIG. 6 a). PcCel6A-S407P andHiCel6A-Y420P also exhibit an increase in optimal temperature whencompared to their respective wild-type (FIGS. 6 b and c).

Increasing the Alkalophilicity of Family 6 Cellulases

The effect of the S413P mutation, alone or in combination with one ormore of G231S, N305S and R410Q, on the pH/activity profile of Family 6cellulase was also studied.

All modified Family 6 cellulases exhibit increased alkalophilicity whencompared to their wild-type. For TrCel6A, the most important shift wasobserved with variants TrCel6A-G231S-R410Q-S413P (+1.25 pH units)followed by TrCel6A-G231S-N305S-R410Q-S413P (+1.01 pH units).

Cellulase systems comprising modified Family 6 cellulases in combinationwith non-Family 6 cellulases show improved thermostability. ATrichoderma cellulase system comprising TrCel6A-S413P maintains at least80% of its maximal activity after incubation in the absence of substrateat 50° C. for 96 hours, while the corresponding cellulase systemcomprising the parent TrCel6A maintains only 50% of its maximal activity(FIG. 8).

In summary, improved thermostable, alkalophilic and/or thermophlicmutant Family 6 cellulase of the invention comprise a proline residue atposition 413 and may further comprise one or more than one of thefollowing amino acid substitutions:

-   -   (i) a substituted amino acid at position 231 such as a polar        amino acid, including, but not limited to, Ser;    -   (ii) a substituted amino acid at position 305, such as a polar        amino acid, including, but not limited to, Ser;    -   (iii) a substituted amino acid at position 410, such as a polar        amino acid, including, but not limited to, Gln; and    -   (iv) combinations of any of the above mutations set out in (i)        to (iii).

Non-limiting examples of preferred Family 6 cellulase mutants comprisinga S413P in combination with the amino acid substitutions listed aboveare given in Table 2.

Furthermore, the modified Family 6 cellulase of the present inventionmay comprise amino acid substitutions not listed above in combinationwith S413P.

The above description is not intended to limit the claimed invention inany manner. Furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

EXAMPLES

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only and should not be used to limit the scope ofthe present invention in any manner.

Examples

Example 1 describes the strains and vectors used in the followingexamples. Examples 2-5 describe the random mutagenesis of the TrCel6Agene, cloning of the random mutagenesis libraries in yeast vectors andhigh-throughput screening to identify modified Family 6 cellulases withincreased thermostability. Examples 6-8 describe the cloning,recombination and expression of the modified and native Family 6cellulase genes in an alternative yeast vector for higher expression.Example 9 describes the enzymatic characterization of modified Family 6cellulases. Example 10 describes genetic constructs to express andsecrete the modified Family 6 cellulases in a filamentous fungus.Example 11 describes the transformation of fungal protoplasts withgenetic constructs expressing modified Family 6 cellulases. Example 12describes the production of modified Family 6 cellulases from modifiedmicrobes in submerged liquid cultures. Example 13 describes thecharacterization of whole Trichoderma cellulases comprising modifiedFamily 6 cellulases in combination with cellulases from other Families.

Example 1 Strains and Vectors

Saccharomyces cerevisiae strain DBY747 (his3-Δ1 leu2-3 leu2-112 ura3-52trp1-289 (amber mutation) gal(s) CUP(r)) was obtained from the ATCC. S.cerevisiae strain BJ3505 (pep4::HIS3 prb-Δ10.6R HIS3 lys2-208 trp1-Δ101ura3-52 gal2 can1) was obtained from Sigma and was a part of theAmino-Terminal Yeast FLAG Expression Kit.

A strain of Trichoderma reesei obtained derived from RutC30 (ATCC#56765; Montenecourt, B. and Eveleigh D. 1979. Adv. Chem. Ser. 181:289-301) comprising a disrupted native TrCel6A gene was used in theexperiments described herein.

Escherichia coli strains HB101 (F⁻ thi-1 hsdS20 (r_(B) ⁻, m_(B) ⁻)supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 rpsL20 (str^(I)) xyl-5mtl-1) and DH5α (F⁻φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r_(k) ⁻, m_(k) ⁺) phoA supE44 thi-1 gyrA96 relA1 λ⁻) were obtained fromInvitrogen.

Humicola insolens and Phanerochaete chrysosporium strains were obtainedfrom ATCC® (#22082™ and #201542™ respectively).

The YEp352/PGK91-1 vector was obtained from the National Institute ofHealth. The YEpFLAG-1 vector was obtained from Sigma as a part of theAmino-Terminal Yeast FLAG Expression Kit. The pALTER®-1 vector wasobtained from Promega as a part of the Altered Site® II in vitromutagenesis System. The pBluescript® II KS-vector was obtained fromStratagene.

Example 2 Cloning of the TrCel6A gene into the YEp352/PGK91-1 andTransformation in Yeast

2.1 Isolation of total RNA from T. reesei and Generation of Total cDNA.

T. reesei biomass was grown under inducing conditions as described inexample 13 then 50 mg of biomass was used to isolate total RNA with theAbsolutely RNA® Miniprep Kit (Stratagene) according to the manufacturerprocedure. Total cDNA was generated from the total RNA using theSuperScript™II Reverse Transcriptase (Invitrogen) according to themanufacturer procedure.

2.2 Cloning and Transformation in Yeast.

In order to facilitate cloning using NheI and KpnI restriction enzymes,the unique NheI site at position 1936 of the YEp352/PGK91-1 vector wasblunted using the DNA Polymerase I large (Klenow) fragment to generateYEp352/PGK91-1ΔNheI.

The cbh2 gene encoding TrCel6A was amplified by PCR from total cDNA(generated as described in example 2.1) using primers (C2STU 5 andC2STU3 that introduce StuI-NheI sites upstream and a KpnI-BglII-StuIsites downstream to the coding sequence. In parallel, the secretionsignal peptide of the TrXyl11B gene was amplified by PCR from a genomicclone of TrXyl11B (pXYN2K2, example 11.3) using primers to introduceBglII at the 5′ end and an NheI site at 3′ end of the amplicon, whichwas subsequently cloned using these restriction sites into pBluescript®II KS-(Stratagene) to generate the plasmid pXYNSS-Nhe. The amplicon wasthen cloned into the unique NheI and Bgl II sites of pXYNSS-Nhe. Afragment comprising the TrCel6A gene operably linked to the secretionsignal peptide of TrXyl11B with BglII sites at the 5′ and 3′ ends wassubsequently amplified by PCR from this intermediate construction usingprimers (BGL2XYF and C2STU3). This amplicon was cloned in the BglII siteof the YEp352/PGK91-1ΔNheI vector to yield to theYEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2 vector (FIG. 2 a) and transformed inyeast strain DBY747 using the procedure described by Gietz, R. D. andWoods, R. A. (Gietz, R. D. and Woods, R. A. 2002. Meth. Enzym. 350:87-96) and plated on SC-Ura plate. Primer sequences are listed below:

      StuI    NheI C2STU5: 5′GAT AGG CCT GCT AGC TGC TCA AGC GTC TGG GGC(SEQ ID NO: 24)         StuI   BglII   KpnI C2STU3: 5′ATCAGG CCT AGA TCT GGT ACC TTA CAG GAA CGA TGG (SEQ ID NO: 25)        BglII BGL2XYF: 5′GAT CAG ATC TAT GGT CTC CTT CAC CTC CCT C (SEQID NO: 26)SC-Ura pate contains:

Component g/L Yeast Nitrogen Base without amino 1.7 acid and ammoniumsulfalte (BD) (NH₄)₂SO₄ (Sigma) 5.0 Complete Supplement Media withouturidine (Clontech) 0.77 Agar (BD) 17.0 Glucose (Fisher) 20.0 pH 5.6

Example 3 Making Error Prone-PCR Libraries of cbh2

Random mutagenesis libraries were generated using two methods: aMn²⁺/dITP method and a biased nucleotides method. For the Mn²⁺/dITPmethod, the TrCel6A gene was amplified fromYEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2 vector using the above-mentionedC2STU3 and BGL2XYF primers in a two step PCR method. In the first step,the amplification occurs for 20 cycles in the presence 20 μM MnCl₂. Thesecond step is done with the same primers but using the product from thefirst step as template and with 0, 25, 50, 75 or 100 μM dITP (0 μM beinga control). For the biased nucleotides method, the PCR is conducted with1:3, 1:5 or 1:10 molar ratio between purine bases and pyrimidine basesrespectively.

To get mostly mutations in the core of the enzyme, the final amplicon inboth cases was cloned using the XhoI and KpnI restriction sites in theYEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2 vector (XhoI cuts right after sequencecoding for S55's codon in the linker of the enzyme) and transformed inS. cerevisiae strain DBY747.

Example 4 Making Site-Directed Semi-Random Libraries of TrCel6A

Glycine residues have no β-carbon and thus have considerably greaterbackbone conformational freedom. By analyzing the three-dimensionalstructure of TrCel6A, 4 glycines residues were targeted to decrease thisdegree of freedom, namely G90, G85, G231 and G384. All but G231positions were saturated and G231 was randomly mutated for an alanine, aproline, a serine or a threonine by megaprimer PCR using the followingprimers:

G⁹⁰ to Xxx: (SEQ ID NO:27) 5′ CCA ACA AAA GGG TTN NNT GAA TAC GTA GCG GG⁸⁵ to Xxx: (SEQ ID NO:28) 5′ CCC AAG GAG TGA CNN NAA CAA AAG GGT TGG²³¹ to A/P/S/T: (SEQ ID NO:29) 5′ GGT GAC CAA CCT CNC NAC TCC AAA GTGTG G³⁸⁴ to Xxx: (SEQ ID NO:30) 5′ CCG CAA ACA CTN NNG ACT CGT TGC TG

All amplicons were cloned in the YEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2vector as described in example 3.

Example 5 Screening of TrCel6A Gene Libraries for Modified Family 6Cellulases with Increased Thermostability

A total of 3371 TrCel6A variants generated as per Example 3 and 4 werescreened as follows: each yeast colony was cultured in a well of a96-deep well plate containing 1 mL of YPD (1% yeast extract, 2% peptone,2% glucose) media and one 1.5 mm glass bead for 2 days in a Vortempapparatus (Labnet) at 650 rpm and at 30° C. The plate was centrifuged at3,000×g for 5 minutes then 300 μL of supernatant was filtered througheach of two Biodyne B positively charged nylon membranes (Pall Gelman)using a Bio-Dot apparatus (Bio-Rad).

Membranes were placed on a moist (not wet) Whatman paper containing 50mM sodium citrate at pH 4.8. One was incubated for 12 minutes at 62° C.and the other one at room temperature (control). Membranes were thenplaced on agar plates containing β-glucan substrate and incubatedovernight at 50° C. in a humidity chamber:

Component g/L (NH₄)₂SO₄ (Sigma) 5.0 β-glucan (Barley, Medium Viscosity;Megazyme) 2.0 Agar (BD) 17.0 Glucose (Fisher) 20.0 pH 5.6

Agar plates were then stained 30-60 minutes by covering them with a 0.1%(w/v) Congo Red solution then rinsed 2-3 times with demineralized waterto remove unbound dye and covered with 1M NaCl for 10-15 min. Theclearing zones could be observed and compared between the control andthe plate that was covered with the heat treated membrane. Each platewas scrutinized by at least two people and every positive variant thatappeared to maintain its activity after the 12 min incubation at 62° C.when compared to the wild-type TrCel6A control was considered aspotential positive. Each potential positive clone was produced again inmicroculture to allow observation of the phenotype on an additionaloccasion and to reduce the possibility of false negative.

From that screening, five positive clones were sequenced to identify themutations they carry. Clone E6 contained a S413P mutation, clones G3 andF7 both contained a G231S mutation, clone A3 contained a N305S mutationand clone 7 contained a R410Q mutation as well as a G82E mutation at theend of the linker peptide.

Example 6 Cloning Modified TrCel6A Genes into the YEpFLAG-1 Vector forHigher Expression from Saccharomyces cerevisiae

In order to facilitate cloning of the modified TrCel6A genes identifiedin Example 5 into the YEpPLAG-1 vector in such a way as to operablinglink the genes to the αmating factor secretion signal peptide, twomodifications were necessary. First, the unique KpnI site present in theα secretion signal peptide sequence (bp 1457) of the YEpFLAG-1 vectorwas removed. This was done by PCR using two complementary mutagenicprimers (5′-FLAGΔKpnI and 3′-FLAGΔKpnI). The mutagenesis reaction wasthen digested with DpnI for 1 hour at 37° C. and the plasmid was allowedto recircularize by placing the tube in boiling water and allowed tocool slowly to room temperature. This reaction was transformed directlyin E. coli DH5α chemically competent cells. A clone that was digestedonly once with KpnI was sequenced to confirm the desired mutation andwas used for further work and named YEpFLAGΔKpn. Primer sequences arelisted below:

                 ΔKpnI 5′-FLAGΔKpnI: 5′CTA AAG AAG AAG GGG TAC ATT TGGATA AAA GAG AC (SEQ ID NO:31)                          66 KpnI3′-FLAGΔKpnI: 5′GTC TCT TTT ATC CAA ATG TAC CCC TTC TTC TTT AG (SEQ IDNO:32)

Second, the T. reesei cbh1 gene was amplified from pCOR132 (Example11.2) by PCR using primers to introduce XhoI-NheI sites at the 5′ endand Kpn1-Apa1 sites at 3′ end of the amplified fragment. This fragmentwas then inserted as an XhoI/ApaI fragments into the XhoI/ApaIlinearized YEpFLAG-1 expression vector. The resulting vector,YEpFLAGΔKpn10, allows insertion of the modified TrCel6A genes identifiedin Example 5 as NheI/KpnI fragments in such a way that the codingregions are operably linked to the α secretion signal peptide.

The YEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2 vectors containing native ormodified TrCel6A genes were isolated from transformants of yeast strainDBY747 using method modified from Hoffman and Winston (Hoffman, C. S.,and Winston, F. 1987. Gene 57: 267-272) and transformed in E. coli HB101chemically competent cells. The modified TrCel6A genes were removed fromthe YEp352/PGK91-1ΔNheI-xyl_(SS)-cbh2 vectors by digestion with NheI andKpnI and cloned in the YEpFLAGΔKpn10 using the same restriction enzymes.The final constructs, YEpFLAGΔKpn10-cbh2, YEpFLAGΔKpn10-G82E-R410Q,YEpFLAGΔKpn10-N305S YEpFLAGΔKpn10-S413P and YEpFLAGΔKpn10-G231S (FIG. 2b), were transformed into yeast strain BJ3505 using the proceduredescribed by Gietz and Woods (Gietz R. D. and Woods R. A. 2002. Meth.Enzym. 350: 87-96) and plated on SC-trp plate. The integrity of thecloned region of all variants was confirmed by DNA sequence analysis.The amino acid sequence of the parent TrCel6A produced by this yeastvector (SEQ ID NO. 23) shows the C-terminal extension containing theFLAG peptide. However, it was determined experimentally that this smallpeptide extension does not in any way contribute to the thermostability,thermophilicity or alkalophilicity of the parent or modified TrCel6Acellulases.

SC-trp Pate Contains:

Component g/L Yeast Nitrogen Base without amino acid and ammonium 1.7sulfalte (BD) (NH₄)₂SO₄ (Sigma) 5.0 Yeast Synthetic Drop-Out MediaSupplement without 1 Tryptophan (Sigma) Agar (BD) 20 Glucose (Fisher) 20

Example 7 Generation of Other TrCel6A Variants, PcCel6A, PcCel6A-S407P,HiCel6A and HiCel6A-Y420P and Their Cloning in the YEpFLAG-1 Vector

7.1 Generation of Other TrCel6A Variants.

TrCel6A variant R410Q-S413P was obtained by error-prone PCR on theTrCel6A-S413P variant while cloned in the YEp352/PGK91-1ΔNheI using theMutazyme® II DNA polymerase (Stratagene). It was then amplified fromthat source using primers 5′FLAG-Cel6A-GR and 3′FLAG-Cel6A-GR thatintroduce sequences homologue to the YEpFLAG-1 vector upstream the NheIsite and downstream the ApaI site respectively.

Mutagenic primers in conjunction with primer 3′FLAG-Cel6A-GR were usedto generate megaprimer PCR of the following TrCel6A mutationcombinations: G231S-S413P, N305S-S413P, G231S-N305S-S413P,G231S-R410Q-S413P, N305S-R410Q-S413P and G231S-N305S-R410Q-S413P. Theresulting PCR products were isolated and used as a reverse primer inconjunction with the forward primer 5′FLAG-Cel6A-GR to generate finalconstructs. Primers sequences are listed below:

5′G231SCBH2 (SEQ ID NO: 35) 5′GGT GAC CAA CCT CTC TAC TCC AAA GTG TG5′N305SGBH2 (SEQ ID NO: 36) 5′CAA TGT CGC CAG CTA CAA CGG G 5′Ce16A-E82G(SEQ ID NO: 38) 5′GTA CCT CCA GTC GGA TCG GGA ACC GCT 5′FLAG-Ce16A-GR(SEQ ID NO:39) 5′AGA GAC TAC AAG GAT GAC GAT GAC AAG GAA TTC CTC GAG GCTAGC TGC TCA AGC G 3′FLAG-Ce16A-GR (SEQ ID NO: 40) 5′GAC CCA TCA GCG GCCGCT TAC CGC GGG TCG ACG GGC CCG GTA CCT TAC AGG AAC G7.2 Generation PcCel6A and PcCel6A-S407P.

Lyophilized P. chrysosporium was resuspended in 300 μL sterile H₂O and50 μL were spreaded onto PDA plates. Plates were incubated at 24° C. for4 days. Spores for P. chrysosporium were inoculated on a cellophanecircle on top of a PDA plate and biomass was harvested after 4-6 days at24° C. Then, 50 mg of biomass was used to isolate total RNA with theAbsolutely RNA® Miniprep Kit (Stratagene) according to the manufacturerprocedure. Total cDNA was generated from the total RNA using theSuperScript™II Reverse Transcriptase (Invitrogen) according to themanufacturer procedure. Gene encoding for PcCel6A was amplified from thecDNA using the following primers:

5′PcCe16A-cDNA (SEQ ID NO: 41) 5′CTA TTG CTA GCT CGG AGT GGG GAC AGT GCGGTG GC 3′PcCe16A-cDNA (SEQ ID NO: 42) 5′CTA TTG AAT TCG GTA CCC TAC AGCGGC GGG TTG GCA GCA GAA AC

PCR amplicon was clone in the pGEM®-T Easy vector by TA-cloningfollowing manufacturer's recommendations. The gene encoding for PcCel6Awas then amplified from that source using primers 5′FLAG-PcCel6A-GR and3′FLAG-PcCel6A-GR that introduce sequences homologue to the YEpFLAG-1vector upstream the NheI site and downstream the SstII siterespectively.

Mutagenic primer 5′PcCel6A-S407P in conjunction with primer3′FLAG-PcCel6A-GR was used to generate megaprimer PCR. The resulting PCRproduct was isolated and used as a reverse primer in conjunction withthe forward primer 5′FLAG-PcCel6A-GR to generate final construct.Primers sequences are listed below:

5′FLAG-PcCe16A-GR (SEQ ID NO: 43)5′AAGGATGACGATGACAAGGAATTCCTCGAGGCTAGCTCGGAGTG GGG ACAGTGC3′FLAG-PcCe16A-GR (SEQ ID NO: 44)5′TGGGACGCTCGACGGATCAGCGGCCGCTTACCGCGGCTACAGCG GCG GGTTGGC5′PcCe16A-S407P (SEQ ID NO: 45) 5′CCCCGCTACGACCCTACTTGTTCTCTG7.3 Generation HiCel6A and HiCel6A-Y420P.

Lyophilized H. insolens was resuspended in 300 μL sterile H₂O and 50 μLwas spreaded onto Emerson YPSS pH 7 agar plate (0.4% Yeast extract, 0.1%K₂HPO₄, 0.05% MgSO₄.7H₂O, 1.5% Glucose, 1.5% Agar). Fungus was incubatedfor 6 days at 45° C. then spores were inoculated in Novo media (as perBarbesgaard U.S. Pat. No. 4,435,307): Incubation for 48 hours at 37° C.in 100 mL growth phase media (2.4% CSL, 2.4% Glucose, 0.5% Soy oil, pHadjusted to 5.5, 0.5% CaCO₃), then 6 mL of pre-culture was transferredinto 100 mL production phase media (0.25% NH₄NO₃, 0.56% KH₂PO₄, 0.44%K₂HPO₄, 0.075% MgSO₄.7H₂O, 2% Sigmacell, pH adjusted to 7, 0.25% CaCO₃)and culture was incubated for up to 4 days prior to biomass harvest.Then, 50 mg of biomass was used to isolate total RNA with the AbsolutelyRNA® Miniprep Kit (Stratagene) according to the manufacturer procedure.Total cDNA was generated from the total RNA using the SuperScript™IIReverse Transcriptase (Invitrogen) according to the manufacturerprocedure. Gene encoding for HiCel6A was amplified from the cDNA usingthe following primers:

5′HiCe16A-cDNA (SEQ ID NO: 46) 5′CTA TTG CTA GCT GTG CCC CGA CTT GGG GCCAGT GC 3′HiCe16A-cDNA (SEQ ID NO: 47) 5′CTA TTG AAT TCG GTA CCT CAG AACGGC GGA TTG GCA TTA CGA AG

PCR Amplicon was Clone in the pGEMO-T Easy vector by TA-Cloningfollowing manufacturer's recommendations. The gene encoding for HiCel6Awas then amplified from that source using primers 5′FLAG-HiCel6A-GR and3′FLAG-HiCel6A-GR that introduce sequences homologue to the YEpFLAG-1vector upstream the NheI site and downstream the ApaI site respectively.

Mutagenic primer 5′HiCel6A-Y420P in conjunction with primer3′FLAG-HiCel6A-GR was used to generate megaprimer PCR. The resulting PCRproduct was isolated and used as a reverse primer in conjunction withthe forward primer 5′FLAG-HiCel6A-GR to generate final construct.Primers sequences are listed below:

5′FLAG-HiCe16A-GR (SEQ ID NO: 48)5′AAGGATGACGATGACAAGGAATTCCTCGAGGCTAGCTGTGCCCC GACTTGGGGC3′FLAG-HiCe16A-GR (SEQ ID NO: 49)5′AGCGGCCGCTTACCGCGGGTCGACGGGCCCGGTACCTCAGAACGG CGGATTGGC5′HiCe16A-Y420P (SEQ ID NO: 50) 5′GCCCGCTACGACCCTCACTGCGGTCTC7.4 Cloning of the Other TrCel6A Variants, PcCel6A, PcCel6A-S407P,HiCel6A and HiCel6A-Y420P in YEpFLAG-1 and Transformation in BJ3505.

The YEpFLAGΔKpn10 vector (example 6) was digested with NheI and ApaI andthe empty vector fragment was isolated. This linear fragment and thefinal PCR products generated in example 8.1 and 8.3 were cloned andtransformed simultaneously by in vivo recombination (Butler, T. andAlcalde, M. 2003. In Methods in Molecular Biology, vol. 231: (F. H.Arnold and G. Georgiou, editors), Humana Press Inc. Totowa (New Jersey),pages 17-22).

The YEpFLAGΔKpn10 vector (example 6) was digested with NheI and SstIIand the empty vector fragment was isolated. This linear fragment and thefinal PCR products generated in example 8.2 were cloned and transformedsimultaneously by in vivo recombination.

Example 8 Medium Scale Expression of Native and Modified Family 6Cellulases in Yeast

One isolated colony of BJ3505 yeast containing YEpFLAG-ΔKpn10-cbh2 wasused to inoculate 5 mL of liquid SC-trp in a 20 mL test-tube. After anovernight incubation at 30° C. and 250 rpm, optical density at 600 m wasmeasured and 50 mL of YPEM liquid media in a 250 mL Erlenmeyer flask wasinoculated with the amount of yeast required to get a final OD₆₀₀ of0.045. After 72 h of incubation at 30° C. and 250 rpm, supernatant washarvested with a 5 minutes centrifugation step at 3,000×g. The BJ3505strains expressing the TrCel6A variants, wild-type HiCel6A and variant,wild-type PcCel6A and variant as well as the empty YEpFLAG-1 vector werecultured the same way.

SC-trp liquid media contains the same components as the SC-trp platementioned in example 7 without the agar. YPEM liquid media contains:

Component per Liter Yeast Extract (BD) 10 g Peptone (BD) 5.0 g Glucose(Fisher) 10 g Glycerol (Fisher) 30 mL

Example 9 Characterization of Modified Family 6 Cellulases from YeastCulture Supernatants

9.1 Comparison of the thermophilicity of the Modified TrCel6A with theNative TrCel6A.

The thermophilicity of each enzyme was determined by measuring therelease of reducing sugars from a soluble β-glucan substrate atdifferent temperatures. Specifically, in a 300 μL PCR plate, 50 μL ofcrude supernatant obtained as per Example 9 was mixed with 50 μL ofpre-heated 1% (w/v) β-glucan (Barley, Medium Viscosity; Megazyme) in 55mM sodium citrate pH 5.0 in 10 different columns of a 96-well PCR plate.Mixtures were incubated for 30 min. at 10 different temperatures of agradient (56, 58.1, 59.8, 62.6, 66, 70, 73.4, 76.2, 78.1 and 80° C.)then released reducing sugars were measured as follows: 100 μL of DNSreagent was added to each well and the plate was incubated 20 minutes at95° C.

DNS reagent contains:

Component g/L 3,5-Dinitosalicylic acid (Acros) 10 Sodium hydroxide(Fisher) 10 Phenol (Sigma) 2 Sodium metabisulfate (Fisher) 0.5

Once the temperature decreased below 40° C., 135 μL of each reactionmixture was transferred to individual wells of a 96-well microplatecontaining 65 μL of Rochelle salts (40% Sodium potassium tartrate) ineach well and OD₅₆₀ was measured using a Fluostar Galaxy microplatereader equipped with a 560 nm filter. Blank value was measured bytreating the supernatant from the strain carrying the empty vector thesame way and was subtracted to each value. Then activity was expressedin percentage relatively to the highest value of the four parametersPolynomial fit for each variant except variantTrCel6A-G231S-N305S-R410Q-S413P for which a four parameters Log Normalfit was used (FIG. 6).

All modified Family 6 cellulases shown an improved optimal temperaturewhen compared to their respective wild-type except variantTrCel6A-G231S-N305S-R40Q-S413P which on the other hand exhibits a broadtemperature range with more then 80% of the maximum activity (FIG. 6).Among all TrCel6A variants, TrCel6A-S413P has the higher optimaltemperature at 72.2° C., an increase of 5.6° C. compared to wild-typeTrCel6A (FIG. 6 a). PcCel6A-S407P and HiCel6A-Y420P also exhibit anincrease in optimal temperature when compared to their respectivewild-type (FIGS. 6 b and c).

9.2 Comparison of the Alkalophilicity of the Modified TrCel6A with theNative TrCel6A.

The alkalophilicity of each enzyme was determined by measuring therelease of reducing sugars from a soluble β-glucan substrate atdifferent pH. Specifically, in a 300 μL PCR plate, 50 μL of crudesupernatant obtained as per example 9 was mixed to 50 μL of pre-heated1% (w/v) β-glucan (Barley, Medium Viscosity; Megazyme) in 55 mM sodiumcitrate, 55 mM sodium phosphate pH 3.0, 4.0, 5.0, 5.75, 6.25, 6.75, 7.25or 8.5 in 8 different columns of the plate (once mixed to thesupernatant and heated at 60-65° C., pHs where 3.95, 4.65, 5.65, 6.25,6.65, 6.95, 7.15 and 7.45 respectively). Mixtures were incubated 30 min.at 65° C. (60° C. for PcCel6A and variant) then released reducing sugarswere measured as per Example 10.1. Background activity from the yeasthost was measured by treating the supernatant from the strain carryingthe empty vector the same way and this activity was subtracted from theactivity value for each variant. Then activity was expressed inpercentage relatively to the highest value of the three parametermechanistic fit for each variant (FIG. 7).

All modified Family 6 cellulases exhibit increased alkalophilicity whencompared to their wild-type. For TrCel6A, the most important shift wasobserved with variants TrCel6A-G231S-R410Q-S413P (+1.25 pH units)followed by TrCel6A-G231S-N305S-R410Q-S413P (+1.01 pH units).

9.3 Comparison of the Thermostability of the Modified TrCel6A with theNative TrCel6A.

The thermostability of each enzyme was determined by measuring therelease of reducing sugars from a soluble β-glucan substrate afterdifferent pre-incubation time of the supernatant at 60° C. (55° C. forPcCel6A and variant). Specifically, in a 300 μL PCR plate, 50 μL ofcrude supernatant obtained as per Example 9 was incubated at 60° C. (55°C. for PcCel6A and variant) for 0, 15, 30, 45, 60, 75, 90 and 120minutes. Then, 50 μL of 1% (w/v) β-glucan (Barley, Medium Viscosity;Megazyme) in 55 mM sodium citrate pH 5.0 was added and mixtures wereincubated 30 minutes 65° C. (60° C. for PcCel6A and variant). Releasedreducing sugars were then measured as per Example 10.1. Backgroundactivity from the yeast host was measured by treating the supernatantfrom the strain carrying the empty vector the same way and this activitywas subtracted from the activity value for each variant. Finally,activity was expressed in percentage relatively to the highest value ofthe three parameter single exponential decay fit for each variant (FIG.5).

All TrCel6A variants have shown increased thermostability when comparedto wild-type TrCel6A (FIG. 5 a). The S413P mutation results in asignificant increase in the thermostability TrCel6A. TrCel6A-S413Pretains 45% of its activity after 60 minutes at 60° C. whereas TrCel6Aretains only 4% of its activity after 60 minutes. This represents agreater improvement in thermostability compared to that of theTrCel6A-S413Y variant disclosed in US Patent Publication No.20060205042, which retained on average 41% of its activity under similarconditions. The highest improvement was observed withTrCel6A-R410Q-S413P and TrCel6A-G231S-R410Q-S413P as both retain 58 and60% of their activity after 60 minutes at 60° C. respectively. Similarlyto TrCel6A, PcCel6A-S407P retains 38% of its activity after 60 minutesat 55° C. whereas PcCel6A retains only 6% of its activity after 60minutes (FIG. 5 b). This supports the claim for which a proline at theequivalent position of TrCel6A residue 413 increases thermostability.

9.4 Comparison of the T₅₀ of the Modified TrCel6A with the NativeTrCel6A.

T₅₀ herein is defined as the temperature at which the crude yeastsupernatant retains 50% of its β-glucan hydrolyzing activity after 15minutes of incubation without substrate. It was determined by measuringthe release of reducing sugars from a soluble β-glucan substrate after15 minutes of pre-incubation at different temperatures. Specifically, ina 300 μL PCR plate, 50 μL of crude supernatant obtained as per Example9, was incubated at 45, 49.2, 53.9, 57.7, 59.5, 60.4, 62.5, 64.2, 66.4,68.9, 72.7 or 75° C. for 15 minutes. Then, 50 μL of 1% (w/v) β-glucan(Barley, Medium Viscosity; Megazyme) in 55 mM sodium citrate pH 5.0 wasadded and mixtures were incubated 30 minutes 65° C. (60° C. for PcCel6Aand variant). Released reducing sugars were then measured as per Example10.1. Background activity from the yeast host was measured by treatingthe supernatant from the strain carrying the empty vector the same wayand this activity was subtracted from the activity value for eachvariant. Finally, activity was expressed in percentage relatively to thehighest value of the four parameter sigmoid fit for each variant (FIG.4).

The T₅₀ of the TrCel6A-S413P was determined to be 64.1° C., as comparedto 59° C. for the parent TrCel6A (FIG. 4 a). This represents an increasein the thermostability by over 5° C. through the introduction of theS413P mutation. This represents a significant improvement in enzymestability compared to the S413Y mutation disclosed US Patent PublicationNo. 20060205042, which shows a very modest 0.2-0.3° C. increase in theTm of the TrCel6A-S413Y over TrCel6A. Although the methods to determinethe Tm disclosed US Patent Publication No. 20060205042 is different fromthe determination of T50 disclosed herein, both methods seek todetermine the temperature at which the protein undergoes a significantand structural change that leads to irreversible inactivation. The T₅₀of the other TrCel6A variants was at least 3.2° C. higher then wild-typeTrCel6A.

PcCel6A-S407P and Hicel6A-Y420P also have shown an increase in TSO whencompared to their respective parent enzyme (FIGS. 4 b and c). This alsosupports the claim for which a proline at the equivalent position ofTrCel6A residue 413 increases thermostability in Family 6 cellulases.

Example 10 Making Genetic Constructs Comprising Modified Family 6Cellulase DNA Sequences

10.1 Isolation of Trichoderma reesei Genomic DNA and Construction of T.reesei Genomic Libraries

A strain of Trichoderma reesei obtained derived from RutC30 (ATCC#56765; Montenecourt, B. and Eveleigh. D. 1979. Adv. Chem. Ser. 181:289-301) comprising a disrupted native TrCel6A gene was used. RutC30 isderived from Trichoderma reesei Qm6A (ATCC # 13631; Mandels, M. andReese, E. T. 1957. J. Bacteriol. 73: 269-278). It is well understood bythose skilled in the art that the procedures described herein, thegenetic constructs from these strains, and the expression of the geneticconstructs in these strains are applicable to all Trichoderma strainsderived from Qm6A.

To isolate genomic DNA, 50 mL of Potato Dextrose Broth (Difco) wasinoculated with T. reesei spores collected from a Potato Dextrose Agarplate with a sterile inoculation loop. The cultures were shaken at 200rpm for 2-3 days at 28° C. The mycelia was filtered onto a GFA glassmicrofibre filter (Whatman) and washed with cold, deionized water. Thefungal cakes were frozen in liquid nitrogen crushed into a powder with apre-chilled mortar and pestle; 0.5 g of powdered biomass was resuspendedin 5 mL of 100 mM Tris, 50 mM EDTA, pH 7.5 plus 1% sodium dodecylsulphate (SDS). The lysate was centrifuged (5000 g for 20 min, 4° C.) topellet cell debris. The supernatant was extracted with 1 volume buffer(10 mM Tris, 1 mM EDTA, pH 8.0) saturated phenol followed by extractionwith 1 volume of buffer-saturated phenol:chloroform:isoamyl alcohol(25:24:1) in order to remove soluble proteins. DNA was precipitated fromthe solution by adding 0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5volumes of cold 95% ethanol. After incubating for at least 1 h at −20°C., the DNA was pelleted by centrifugation (5000 g for 20 min, 4° C.),rinsed with 10 mL 70% ethanol, air-dried and resuspended in 1 mL 10 mMTris, 1 mM EDTA, pH 8.0. RNA was digested by the addition ofRibonuclease A (Roche Diagnostics) added to a final concentration of 0.1mg/mL and incubation at 37° C. for 1 hour. Sequential extractions with 1volume of buffer-saturated phenol and 1 volume of buffer-saturatedphenol:chloroform:isoamyl alcohol (25:24:1) was used to remove theribonuclease from the DNA solution. The DNA was again precipitated with0.1 volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes of cold 95%ethanol, pelleted by centrifugation, rinsed with 70% ethanol, air-driedand resuspended in 50 μL of 10 mM Tris, 1 mM EDTA, pH 8.0. Theconcentration of DNA was determined by measuring the absorbance of thesolution at 260 nm (p. C1 in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press 1989, whichis incorporated herein by reference, and hereafter referred to asSambrook et al.).

Two plasmid libraries and one phage library were constructed usinggenomic DNA isolated from T. reesei strain M2C38. The plasmid librarieswere constructed in the vector pUC119 (Viera and Messing, MethodsEnzymol. 153:3, 1987) as follows: 10 μg genomic DNA was digested for 20hrs at 37° C. in a 100 μL volume with 2 units/μg of BamH1 or EcoR1restriction enzymes. The digested DNA was fractionated on a 0.75%agarose gel run in 0.04 M Tris-acetate, 1 mM EDTA and stained withethidium bromide. Gel slices corresponding to the sizes of the genes ofinterest (based on published information and Southern blots) wereexcised and subjected to electro-elution to recover the DNA fragments(Sambrook et al., pp. 6.28-6.29). These enriched fractions of DNA wereligated into pUC119 in order to create gene libraries in ligationreactions containing 20-50 μg/mL DNA in a 2:1 molar ratio ofvector:insert DNA, 1 mM ATP and 5 units T4 DNA ligase in a total volumeof 10-15 μL at 4° C. for 16 h. Escherichia coli strain HB101 waselectroporated with the ligation reactions using the Cell Porator System(Gibco/BRL) following the manufacturer's protocol and transformantsselected on LB agar containing 70 μg/mL ampicillin.

The phage library was constructed in the vector λDASH (Stratagene, Inc.)as follows: genomic DNA (3 μg) was digested with 2, 1, 0.5 and 0.5units/μg BamHI for 1 hour at 37° C. to generate fragments 9-23 kB insize. The DNA from each digest was purified by extraction with 1 volumeTris-staturated phenol:choroform:isoamyl alcohol (25:24:1), followed byprecipitation with 10 μL 3 M sodium acetate, pH 5.2 and 250 μl 95%ethanol (−20° C.). The digested DNA was pelleted by microcentrifugation,rinsed with 0.5 mL cold 70% ethanol, air-dried and resuspended in 10 μLsterile, deionized water. Enrichment of DNA fragments 9-23 kB in sizewas confirmed by agarose gel electrophoresis (0.8% agarose in 0.04 MTris-acetate, 1 mM EDTA). Digested DNA (0.4 μg) was ligated to 1 μgλDASH arms predigested with BamHI (Stratagene) in a reaction containing2 units T4 DNA ligase and 1 mM ATP in a total volume of 5 μl at 4° C.overnight. The ligation mix was packaged into phage particles using theGigaPack® II Gold packaging extracts (Stratagene) following themanufacturer's protocol. The library was titred using the E. coli hoststrain XL1-Blue MRA (P2) and found to contain 3×10⁵ independent clones.

10.2 Cloning the Cellobiohydrolase I (cbh1) and Cellobiohydrolase II(cbh2) Genes from pUC119 Libraries

E. coli HB101 transformants harboring cbh1 or cbh2 clones fromrecombinant pUC119-BamH1 or -EcoRI libraries were identified by colonylift hybridization: 1−3×10⁴ colonies were transferred onto HyBond™ nylonmembranes (Amersham); membranes were placed colony-side up onto blottingpaper (VWR 238) saturated with 0.5 M NaOH, 1 M NaCl for 5 min to lysethe bacterial cells and denature the DNA; the membranes were thenneutralized by placing them colony-side up onto blotting paper (VWR 238)saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 min; the membraneswere allowed to air-dry for 30 min and the DNA was then fixed to themembranes by baking at 80° C. for 2 h.

³²P-labelled probes were prepared by PCR amplification of short (0.7-1.5kB) fragments of the cbh1 and cbh2 coding regions from the enriched poolof BamH1 or EcoR1 fragments, respectively, in a labelling reactioncontaining 10-50 ng target DNA, and 0.2 mM each of d(GCT)TP, 0.5 μMdATP, 20-40 μCi α-³²P-dATP, 10 pmole oligonucleotide primers and 0.5units Taq polymerase in a total volume of 20 μL. The reaction wassubjected to 6-7 cycles of amplification (95° C., 2 min; 56° C., 1.5min; 70° C., 5 min). The amplified, ³²P-labelled DNA was precipitated bythe addition of 0.5 mL 10% (w/v) trichloroacetic acid and 0.5 mg yeasttRNA. The DNA was pelleted by microcentrifugation, washed twice with 1mL 70% ethanol, air-dried and resuspended in 1 M Tris pH 7.5, 1 mM EDTA.

Nylon membranes onto which the recombinant pUC119 plasmids had beenfixed were prehybridized in heat-sealed bags for 1 h at 60-65° C. in 1 MNaCl, 1% SDS, 50 mM Tris, 1 mM EDTA pH 7.5 with 100 μg/mL denaturedsheared salmon sperm DNA. Hybridizations were performed in heat-sealedbags in the same buffer with only 50 μg/mL denatured sheared salmonsperm DNA and 5×10⁶-5×10⁷ cpm of denatured cbh1 or cbh2 probe for 16-20h at 60-65° C. Membranes were washed once for 15 min with 1 M NaCl, 0.5%SDS at 60° C., twice for 15 min each with 0.3M NaCl, 0.5% SDS at 60° C.and once for 15 min with 0.03M NaCl, 0.5% SDS at 55° C. Membranes wereagain placed in heat-sealed bags and exposed to Kodak RP X-ray film to16-48 h at −70° C. The X-ray film was developed following themanufacturer's protocols. Colonies giving strong or weak signals werepicked and cultured in 2×YT media supplemented with 70 μg/mL ampicillin.Plasmid DNA was isolated from these cultures using the alkaline lysismethod (Sambrook, et al., pp. 1.25-1.28) and analyzed by restrictiondigest, Southern hybridization (Sambrook, et al., pp. 9.38-9.44) and PCRanalysis (Sambrook, et al., pp. 14.18-14,19).

Clones carrying the cbh1 gene were identified by colony lifthybridization of the pUC119-BamH1 library with a 0.7 kb cbh1 probeprepared using oligonucleotide primers designed to amplify bp 597-1361of the published cbh1 sequence (Shoemaker et al., Bio/Technology 1:691-696, 1983; which is incorporated herein by reference). A cbh1 clone,pCOR132, was isolated containing a 5.7 kb BamH1 fragment correspondingto the promoter (4.7 kb) and 1 kb of the cbh1 structural gene (2.3 kb).From this, a 2.5 kb EcoR1 fragment containing the cbh1 promoter (2.1 kb)and 5′ end of the cbh1 coding region (0.4 kb) was subcloned into pUC119to generate pCB152. Clones carrying the cbh2 gene were identified bycolony lift hybridization of the pUC119-EcoR1 library with a 1.5 kb cbh2probe prepared using oligonucleotide primers designed to amplify bp580-2114 of the published cbh2 sequence (Chen et al. Bio/Technology 5:274-278, 1987). A cbh2 clone, pZUK600 was isolated containing a 4.8 kbEcoR1 fragment corresponding to the promoter (600 bp), structural gene(2.3 kb) and terminator (1.9 kb).

10.3 Cloning Xylanase II (xln2) gene from λDASH Libraries

Digoxigen-11-dUTP labelled probes were prepared from PCR amplifiedcoding regions of the xln2 gene by random prime labeling using the DIGLabeling and Detection kit (Roche Diagnostics) and following themanufacturer's protocols. Genomic clones containing the xln2 gene wereidentified by plaque-lift hybridization of the λDASH library. For eachgene of interest, 1×10⁴ clones were transferred to Nytran® (Schleicherand Schull) nylon membranes. The phage particles were lysed and thephage DNA denatured by placing the membranes plaque-side up on blottingpaper (VWR238) saturated with 0.5 M NaOH, 1 M NaCl for 5 min. Themembranes were then neutralized by placing them plaque-side up ontoblotting paper saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 minand subsequently allowed to air-dry for 30 min. The DNA was then fixedto the membranes by baking at 80° C. for 2 h. The membranes wereprehybridized in heat-sealed bags in a solution of 6× SSPE, 5×Denhardt's, 1% SDS plus 100 μg/mL denatured, sheared salmon sperm DNA at65° C. for 2 h. The membranes were then hybridized in heat-sealed bagsin the same solution containing 50 μg/mL denatured, sheared salmon spermDNA and 0.5 μg of digoxigen-dUTP labelled probes at 65° C. overnight.The membranes were washed twice for 15 min in 2× SSPE, 0.1% SDS at RT,twice for 15 min in 0.2× SSPE, 0.1% SDS at 65° C. and once for 5 min in2× SSPE. Positively hybridizing clones were identified by reaction withan anti-digoxigenin/alkaline phosphatase antibody conjugate,5-bromo-4-chloro-3-indoyl phosphate and 4-nitro blue tetrazoliumchloride (Roche Diagnostics) following the manufacturer's protocol.Positively hybridizing clones were further purified by a second round ofscreening with the digoxigen-dUTP labelled probes.

Individual clones were isolated and the phage DNA purified as describedin Sambrook et al. pp. 2.118-2.121 with the exception that the CsClgradient step was replaced by extraction with 1 volume ofphenol:choroform:isoamyl alcohol (25:24:1) and 1 volume ofchloroform:isoamyl alcohol (24:1). The DNA was precipitated with 0.1volumes of 3 M sodium acetate, pH 5.2 and 2.5 volumes cold 95% ethanol.The precipitated phage DNA was washed with 0.5 mL cold 70% ethanol,air-dried and resuspended in 50 μL 10 mM Tris, 1 mM EDTA pH 8.0.Restriction fragments containing the genes of interest were identifiedby restriction digests of the purified phage DNA and Southern blothybridization (Sambrook, et al., pp. 9.38-9.44) using the samedigoxigen-dUTP labelled probes used to screen the λDASH library. Themembranes were hybridized and positively hybridizing fragmentsvisualized by the same methods used for the plaque lifts. Once thedesired restriction fragments from each λDASH clone were identified, therestriction digests were repeated, the fragments were resolved on a 0.8%agarose gel in TAE and the desired bands excised. The DNA was elutedfrom the gel slices using the Sephaglas B and Prep Kit (Pharmacia)following the manufacturer's protocol.

Clones carrying the xln2 gene were identified by colony lifthybridization of the λDASH library (Example 7) with a xln2 probecomprising bp 100-783 of the published xln2 sequence (Saarelainen etal., Mol. Gen. Genet. 241: 497-503, 1993). A 5.7 kb Kpn1 fragmentcontaining the promoter (2.3 kb), coding region (0.8 kb) and terminator(2.6 kb) the xln2 gene was isolated by restriction digestion of phageDNA purified from a λDASH xln2 clone. This fragment was subcloned intothe Kpn1 site of pUC119 to generate the plasmid pXYN2K-2.

10.4: Construction of a Vector Directing the Expression of ModifiedFamily 6 Cellulase in Trichoderma reesei.

A 2.3 kb fragment containing the promoter and secretion signal of thexln2 gene (bp −2150 to +99 where +1 indicates the ATG start codon and+97-99 represent the first codon after the TrXyl11 secretion signalpeptide) was amplified with Pwo polymerase from the genomic xln2subclone pXYN2K-2 (Example 7) using an xln2-specific primer containing aNheI directly downstream of the Gln at codon 33 and the pUC reverseprimer (Cat. No. 18432-013, Gibco/BRL) which anneals downstream of theKpn1 site at the 5′ end of the xln2 gene. This xlz2 PCR product wasinserted as a blunt-ended fragment into the SmaI site of the pUC119polylinker in such an orientation that the BamHI site of the polylinkeris 3′ to the NheI site; this generated the plasmid pUC/XynPSS(Nhe). Thesame xln2 PCR product was reisolated from pUC/XynPSS(Nhe) by digestionwith EcoRI (which was amplified as part of the pUC119 polylinker frompXYN2K-2) and BamHI and inserted into the plasmid pBR322L (a derivativeof pBR322 containing an Sph1-Not1-Sal1 adaptor between the original Sph1and Sal1 sites at bp 565 and 650), also digested with EcoRI and BamHI,to generate the plasmid pBR322LXN. To facilitate high level expressionof the modified xylanases, a 1.3 kb HindIII fragment comprising bp −1400to −121 of the xln2 promoter in pBR322LXN was replaced with a 1.2 kbHindIII fragment comprising bp −1399 to −204 of the cbh1 promoter whichwas isolated by HindIII digestion of pCOR132; this generated the plasmidpBR322LC/XN. Finally, the EcoR1 site of pBR322LXC was then blunted withKlenow and Spe1 linkers (Cat. No. 1086, New England Biolabs) were addedto generate pBR322SpXC.

A fragment containing the TrCel6A-S413P gene was isolated from theYEpFLAGΔKpn10-cbh2 vector (described in Example 6 above) by digestionwith NheI and KpnI inserted into pCB219N-N digested with NheI and BamHIto generate pS413P/C2ter. To make pCB219N-N, a cbh2 terminator fragmentwas amplified from the pZUK600 (described in Example 7, above) templateusing a primer homologous to bp 2226-2242 of the published 3′untranslated region of the cbh2 gene (Chen et al., 1987) containing ashort polylinker comprising XbaI-NheI-BamHI-SmaI-KpnI sites at the 5′end and the pUC forward primer (Cat. No. 1224, New England Biolabs)which anneals upstream of the EcoR1 site at the 3′ end of cbh2 inpZUK600. This fragment was digested at the engineered XbaI and EcoRIsites and inserted into the corresponding sites of pUC119 to generatepCB219. An EcoR1-Not1 adaptor (Cat. No. 35310-010, Gibco/BRL) wasinserted into the unique EcoR1 site of pCB219 to generate pCB219N. Afragment comprising the TrCel6A gene and the cbh2 terminator wasisolated from pS413P/C2ter by digestion with NheI and NotI and insertedinto pBR322SpXC digested with NheI and NotI to generate the expressioncassette pc/xS413P-EC.

The selection cassette containing plasmid, pNCBgINSNB(r), was derivedfrom a N. crassa pyr4 containing plasmid, pFB6 (Radford, A., Buston, F.P., Newbury, S. F. and Glazebrook, J. A. (1985) Regulation of pyrimidinemetabolism in Neurospora. In Molecular Genetics of Filamentous Fungi(Timberlake, W. E., editor), Alan R. Liss (New York), pages 127-143). A3.2 kb BglII fragment from pFB6 containing the N. crassa pyr4 gene(GenBank accession M13448) as well as its promoter, terminator and some5′ UTR sequences was cloned into the BamHI site of pUC119 modified tocontain NotI, SmaI, NheI and BglII sites in the polylinker (betweenEcoRI and SacI) to generate pNCBgl-NSNB(r). An SpeI/NotI fragmentcomprising the TrCel6A-S413P gene operably linked to the cbh1 promoter,xln2 secretion signal peptide and cbh2 transcriptional terminator wasisolated from the expression cassette vector pc/xS413P-EC and insertedinto pNCBgl-NSNB(r) digested with NheI (SpeI and NheI having compatible5′ overhanging sequences) and NotI to generate p^(c)/_(x)-S413P-TV. Thisfinal construct was linearized by NotI prior to transformation ofTrichoderma reesei.

Example 11 Transformation of the Trichoderma reesei

11.1 Isolation of pyr4 Auxotrophs

In order to use the N. crassa pyr4 gene as a selectable marker, aspontaneous pyr4 auxotroph was isolated as follows: 1×10⁶ spores of T.reesei were plated onto minimal media containing 5 mM uridine and 0.15%(w/v) of the uridine analog 5-fluoroorotic acid (FOA) as previouslydescribed for the selection of pyr4 auxotrophs of T. reesei (Berges, T.and Barreau, C. 1991 Curr Genet. 19(5):359-65). The ability to grow onFOA-containing media will allow for selection of mutants disrupted ineither the pyr2 gene encoding orotate phosphoribosyl transferase or thepyr4 gene encoding orotidine 5′-phosphate decarboxylase. SpontaneousFOA-resistant colonies were subjected to secondary selection of minimalmedia with and without uridine. Spores of FOA-resistant colonies thatcould not grow on minimal media were then transformed with pNCBglNSNB(r)(described in Example 11.4) and selected for growth on minimal media.Only those strains that were complemented by the N. crassa pyr4 gene inpNCBgINSNB(r) will grow on minimal media and are true pyr4 auxotrophs.Using these procedures, a stable pyr4 auxotroph of T. reesei wasobtained.

11.2 Transformation of Protoplasts of T. reesei pyr4 Auxotrophs.

5×10⁶ spores of T. reesei were plated onto sterile cellophane on PotatoDextrose agar supplemented with 5 mM uridine and are incubated for 20hours at 30° C. to facilitate spore germination and mycelial growth.Cellophane discs with mycelia were transferred to 10 mL of aprotoplasting solution containing 7.5 g/L Driselase and 125 units ofprotease free β-glucanase (InterSpex Products Inc., Cat. Nos. 0465-1 and0410-3, respectively) in 50 mM potassium phosphate buffer, pH 6.5containing 0.6 M ammonium sulfate (Buffer P). The mycelial mat wasdigested for 5 hours with shaking at 60 rpm. Protoplasts were separatedfrom undigested mycelia by filtration through sterile No. 30 MIRACLOTH™and collected into a sterile 50 mL round-bottom centrifuge tube andrecovered by centrifugation at 1000-1500×g for 10 min at roomtemperature. Protoplasts were washed with 5 mL of Buffer P andcentrifuged again at 1000-1500×g for 10 min at room temperature.Protoplasts were resuspended in 1 mL of STC buffer (1.2 M sorbitol, 10mM CaCl₂, 10 mM Tris-HCL, pH 7.5). For transformation, 0.1 mL ofresuspended protoplasts were combined with 10 μg of vector DNA and 25 μLof PEG solution (25% PEG 4000, 50 mM CaCl₂, 10 mM Tris-HCl, pH 7.5).After incubation in an ice water bath for 30 min, 1 mL of PEG solutionwas added and the mixture incubated for 5 min at room temperature.Transformation mix was diluted with 2 mL of 1.2 M sorbitol in PEGsolution and the entire mix was added to 25 mL of molten MMSS agar media(see below) cooled to about 47° C. and the protoplast suspension pouredover MMSS agar. Plates were incubated at 30° C. until colony growth wasvisible. Transformants were transferred to individual plates containingMM agar and allowed to sporulate. Spores were collected and plated athigh dilution on MM agar to isolate homokaryon transformants, which werethen plated onto PDA to allow for growth and sufficient sporulation toinoculate the screening cultures as described in Example 13 below.

Minimal medium (MM) agar contains the following components:

Reagent Per L KH₂PO₄ 10 g (NH₄)₂SO₄ 6 g Na₃Citrate•2H₂O 3 g FeSO₄•7H₂O 5mg MnSO₄•H₂O 1.6 mg ZnSO₄•7H₂O 1.4 mg CaCl₂•2H₂O 2 mg Agar 20 g 20%Glucose f.s. 50 mL 1 M MgSO₄•7H₂O f.s. 4 mL pH to 5.5

MMSS agar contains the same components as MM agar plus 1.2 M sorbitol, 1g/L YNB (Yeast Nitrogen Base w/o Amino Acids from DIFCO Cat. No. 291940)and 0.12 g/L amino acids (-Ura DO Supplement from BD Biosciences Cat.No. 630416).

Example 12 Production of Modified Family 6 Cellulases in Liquid Cultures

Individual colonies of Trichoderma were transferred to PDA plates forthe propagation of each culture. Sporulation was necessary for theuniform inoculation of the micro-cultures used in testing the ability ofthe culture to produce the modified TrCelA. variants with increasedthermostability. The culture media is composed of the following:

Component g/L (NH₄)₂SO₄ 12.7 KH₂PO₄ 8.00 MgSO₄•7H₂O 4.00 CaCl₂•2H₂O 1.02Corn Steeped Liquor 5.00 CaCO₃ 20.00 Carbon source** 30-35 Traceelements* 2 mL/L *Trace elements solution contains 5 g/L FeSO₄.7H₂0; 1.6 g/L MnSO₄.H₂0;1.4 g/L ZnSO₄.7H₂0.** glucose, Solka floc, lactose, cellobiose,sophorose, corn syrup, or Avicel. The carbon source can be sterilizedseparately as an aqueous solution at pH 2 to 7 and added to theremaining media initially or through the course of the fermentation.

Individual transformants were grown in the above media in 1 mL culturesin 24-well micro-plates. The initial pH was 5.5 and the media sterilizedby steam autoclave for 30 minutes at 121° C. prior to inoculation. Forboth native and transformed cells, spores were isolated from the PDAplates, suspended in water and 10⁴-10⁵ spores per mL were used toinoculate each culture. The cultures were shaken at 500 rpm at atemperature of 30° C. for a period of 6 days. The biomass was separatedfrom the filtrate containing the secreted protein by centrifugation at12,000 rpm. The protein concentration was determined using the Bio-RadProtein Assay (Cat. No. 500-0001). Expression of TrCelA-S413P wasdetermined as described in Example 14.

Example 13 Characterization of T. reesei Culture Filtrates ComprisingModified Family 6 Cellulases

The expression of TrCel6A-S413P in culture filtrates of T. reeseitransformants was determined by Western blot hybridization of SDS-PAGEgels. Specifically, equal amounts of total secreted protein in 10-20 μLof culture filtrate from the TrCel6A-S413P transformants, the parentstrain P107B and a strain expressing the native, unmodified TrCel6A(strain BTR213) were added to an equal volume of 2× Laemmli buffer (0.4g SDS/2 mL glycerol/1 mL 1M Tris-HCl, pH 6.8/0.3085 g DTT/2 mL 0.5%bromophenol blue/0.25 mL β-mercaptoethanol/10 mL total volume). 10 uL ofeach prepared sample was resolved on a 10% SDS polyacrylamide gel using24 mM Tris, 192 mM glycine pH 6.8, 10 mM SDS as running buffer. Theseparated proteins were transferred electrophoretically from theacrylamide gel to a PVDF membrane, prewetted with methanol, in 25 mMTris/192 mM glycine buffer containing 20% methanol. The membrane wassubsequently washed in 30 mL of BLOTTO buffer (5% skim milk powder in 50mM Tris-HCl, pH 8.0). The membrane was probed with 30 mL of a 1:20,000dilution of polyclonal antibodies specific to TrCel6A in BLOTTOovernight at room temperature, washed twice more for 10 min with anequal volume of BLOTTO at room temperature, then probed for 1 hour witha 1:3000 dilution of goat anti-rabbit/alkaline phosphatase conjugate inBLOTTO at room temperature. Finally, the membrane was washed twice for15 min each at room temperature with an excess of 50 mM Tris-HCl, pH8.0. Hybridizing complexes containing TrCel6A were visualized bytreatment of the membrane with 10 mL of a 100 mM NaCl/100 mM Tris-HCl,pH 9.5 buffer containing 45 μl of 4-nitro blue tetrazolium chloride(Roche Diagnostics) and 35 μL of 5-bromo-4-chloro-3-indoyl phosphate(Roche Diagnostics) at room temperature until bands were clearlyvisible. Positively hybridizing bands of ˜60 kDa were observed in theculture filtrates from most transformants, the positive control strainBTR213, but not from the culture filtrate from strain P107B.Transformant P474B expresses and secretes approximately the same levelof TrCel6A-S413P as the amount of unmodified TrCel6A expressed andsecreted by the control strain BTR213.

The stability of the Trichoderma cellulases containing TrCel6A orTrCel6A-S413P was assessed incubation of the cellulases in 50 mM citratebuffer, pH 4.8 at 50° C. for up to 96 hours and then measuring theresidual activity of the cellulase by nephelometry (Enari, T. M. andNiku-Paavola M. L. 1988. Meth. Enzym. 160: 117-126).

While the rate of cellulose hydrolysis by the untreated TrCel6A andTrCel6A-S413P cellulases was identical, after incubation for up to 96hours in the absence of substrate, the TrCel6A-S413P cellulasemaintained much higher activity than the TrCel6A cellulase (FIG. 8).Thus, improvements in the thermostability of TrCel6A also improved thethermostability of a whole Trichoderma cellulase system comprising themodified Family 6 cellulase and other components.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

All references and citations are herein incorporated by reference.

REFERENCES

-   Ai, Y. C. and Wilson, D. B. 2002. Enzyme Microb. Technol.    30:804-808.-   Atomi, H. 2005. Curr. Opin. Chem. Biol. 9:166-173.-   Berges, T. and Barreau, C. 1991 Curr Genet. 19(5):359-65-   Bhat, M. K. 2000. Biotechnol. Adv. 18:355-383.-   Butler, T. and Alcalde, M. 2003. In Methods in Molecular Biology,    vol. 231: (F. H. Arnold and G. Georgiou, editors), Humana Press Inc.    Totowa (New Jersey), pages 17-22.-   Chica, R. A., et al. 2005. Curr. Opin. Biotechnol. 16:378-384.-   Claeyssens, M. and Henrissat, B. 1992, Protein Science 1:    1293-1297).-   Claeyssens, M., et al. 1997. Eds.; The Royal Society of Chemistry,    Cambridge.-   Davies, G. J., et al. 2000. Biochem. J. 348:201-207.-   Eijsink, V. G., et al. 2004. J. Biotechnol. 113:105-20.-   Eijsink V G, et al. 2005. Biomol. Eng. 22:21-30.-   Foreman, P. K., et al. 2003. J. Biol. Chem. 278:31988-31997.-   Gietz, R. D. and Woods, R. A. 2002. Meth. Enzym. 350: 87-96.-   Gray, K. A., et al. 2006. Curr. Opin. Chem. Biol. 10:141-146.-   Hoffman, C. S., and Winston, F. 1987. Gene 57: 267-272.-   Hughes, S. R., et al. 2006. Proteome Sci. 4:10-23.-   Lehtio, J., et al. 2003. Proc Natl Acad Sci USA. 100:484-489.-   Li, W. F., et al. 2005 Biotechnol. Adv. 23:271-281.-   Lin, Y. and Tanaka, S. 2006. Appl. Microbiol. Biotechnol.    69:627-642.-   Mathrani, I. and Ahring, B. K. 1992 Appl. Microbiol. Biotechnol.    38:23-27.-   Meinke, A., et al. 1995. J. Biol. Chem. 270:4383-4386.-   Radford, A., et al. 1985. In Molecular Genetics of Filamentous Fungi    (Timberlake, W. E., editor), Alan R. Liss (New York), pages 127-143-   Rouvinen, J., et al. 1990. Science 249:380-386. Erratum in: Science    1990 249:1359.-   Saarelainen, R., et al. 1993. Mol. Gen. Genet. 241: 497-503.-   Sadeghi, M., et al. 2006. Biophys. Chem. 119:256-270.-   Sambrook, et al. 1989. Molecular Cloning: A Laboratory Manual,    Second Edition”, Cold Spring Harbor Press-   Srisodsuk, M., et al. 1993. J. Biol. Chem. 268:20756-20761.-   Spezio, M., et al. 1993. Biochemistry. 32:9906-9916.-   Tomme, P., et al. 1988. Eur. J. Biochem 170:575-581.-   Varrot, A., et al. 2005. J. Biol. Chem. 280:20181-20184.-   Varrot, A., et al. 1999. Biochem. J. 337:297-304.-   Vieille, C. and Zeikus, G. J. 2001. Microbiol. Mol. Biol. Rev.    65:1-43.-   Viera and Messing 1987. Methods Enzymol. 153:3-   von Ossowski, I., et al. 2003. J Mol. Biol. 333:817-829.-   Wohlfahrt, G., et al. 2003. Biochemistry. 42:10095-10103.-   Zhang S, et al. 2000. Eur. J. Biochem. 267:3101-15.

1. An isolated Family 6 cellulase comprising one or more mutations, saidone or more mutations consisting essentially of a non-native prolineresidue at position 413 and optionally with: (i) a substitutednon-native amino acid at position 231 consisting of Ser or Thr; (ii) asubstituted non-native amino acid at position 305 consisting of Ser orThr; (iii) a substituted amino acid at position 410 consisting of Gln orAsn; (iv) a substituted Glu amino acid at position 82; or (v) acombination thereof, said positions being determined from sequencealignment of said isolated Family 6 cellulase with a Trichoderma reeseiCe16A amino acid sequence as defined in SEQ ID NO:1, wherein saidisolated cellulase exhibits enhanced thermostability, alkalophilicity,thermophilicity or a combination thereof relative to a correspondingparent Family 6 cellulase, and wherein the isolated Family 6 cellulaseis derived from a fungal Family 6 cellulase.
 2. The isolated Family 6cellulase of claim 1, comprising the substituted non-native amino acidat position 231 consisting of Ser or Thr.
 3. The isolated Family 6cellulase of claim 1, comprising the substituted non-native amino acidat position 305 consisting of Ser or Thr.
 4. The isolated Family 6cellulase of claim 1, comprising the substituted non-native amino acidat position 410 consisting of Gln or Asn.
 5. The isolated Family 6cellulase of claim 1, comprising the substituted non-native amino acidsat positions 231 , 410 and 305 , wherein the non-native amino acids atpositions 231 and 305 are Ser residues and wherein the substitutednon-native amino acid at position 410 is Gln.
 6. The isolated Family 6cellulase of claim 1, wherein said filamentous fungus is Trichodermareesei.
 7. The isolated Family 6 cellulase of claim 1, wherein saidisolated Family 6 cellulase has an increase in thermostability, asevidenced by an increase in its T₅₀ of at least 5° C., over that of theparent Family 6 cellulase.
 8. The isolated Family 6 cellulase of claim1, wherein said isolated Family 6 cellulase has an increase inthermostability, as evidenced by an increase in its T₅₀ of at least 9°C., over that of the parent Family 6 cellulase.
 9. The isolated Family 6cellulase of claim 1, wherein said isolated Family 6 cellulase has anincrease in thermophilicity, as evidenced by an increase in its T_(opt)of at least 1.5° C., over that of the parent Family 6 cellulase.
 10. Theisolated Family 6 cellulase of claim 1, wherein said isolated Family 6cellulase has an increase in thermophilicity, as evidenced by anincrease in its T_(opt) of at least 2.5° C., over that of the parentFamily 6 cellulase.
 11. The isolated Family 6 cellulase of claim 1,wherein said isolated Family 6 cellulase has an increase inalkalophilicity, as evidenced by an increase in its pH_(opt) of at least0.5 units, over that of the parent Family 6 cellulase.
 12. An isolatedFamily 6 cellulase selected from the group consisting of: (SEQ ID NO:12) TrCe16A-S413P; (SEQ ID NO: 13) TrCe16A-G82E-G231S-N305S-R410Q-S413P;(SEQ ID NO: 14) TrCe16A-G231S-S413P; (SEQ ID NO: 15)TrCe16A-N305S-S413P; (SEQ ID NO: 16) TrCe16A-R410Q-S413P; (SEQ ID NO:17) TrCe16A-G231S-N305S-S413P; (SEQ ID NO: 18)TrCe16A-G231S-R410Q-S413P; (SEQ ID NO: 19) TrCe16A-N305S-R410Q-S413P;(SEQ ID NO: 20) TrCe16A-G231S-N305S-R410Q-S413P; (SEQ ID NO: 21)HiCe16A-Y420P; and (SEQ ID NO: 22) PcCe16A-S407P.


13. The isolated Family 6 cellulase of claim 1, wherein the isolatedFamily 6 cellulase has 64-100% sequence identity to amino acids 82-447of the Trichoderma reesei Ce16A amino acid sequence as defined in SEQ IDNO:1.
 14. An isolated Family 6 cellulase comprising one or moremutations, including a non-native proline residue at position 413, saidposition determined from sequence alignment of said modified cellulasewith a Trichoderma reesei Ce16A amino acid sequence as defined in SEQ IDNO:1, wherein said isolated cellulase is derived from a fungal Family 6cellulase and exhibits enhanced thermostability, alkalophilicity,thermophilicity or a combination thereof relative to a correspondingparental Family 6 cellulase, and wherein said isolated Family 6cellulase has a catalytic domain comprising an active site comprising acentral β-barrel comprising seven parallel β-strands connected by fiveα-helices, a C-terminal loop that forms a tunnel with an N-terminal loopthat encloses the active site, and said one or more mutations thatenhance thermostability, alkophilicity or thermophilicity.
 15. Anisolated Family 6 cellulase comprising one or more mutations, includinga non-native proline residue at position 413, said position determinedfrom sequence alignment of said modified cellulase with a Trichodermareesei Ce16A amino acid sequence as defined in SEQ ID NO:1, wherein saidisolated cellulase is derived from a fungal Family 6 cellulase andexhibits enhanced thermostability, alkalophilicity, thermophilicity or acombination thereof relative to a corresponding parental Family 6cellulase, and wherein said isolated Family 6 cellulase has at least 98%identity to amino acids 82-447 of the Trichoderma reesei Ce16A aminoacid sequence as defined by SEQ ID NO:
 1. 16. A method of treating acellulosic substrate, comprising contacting said cellulosic substratewith the isolated Family 6 cellulase of any one of claims 1-5, 6, 7-12,13, 14 or
 15. 17. A process for producing an isolated Family 6 cellulaseof any one of claims 1-5, 6, 7-12, 13, 14 or 15, the process comprising(i) transforming fungal host cells with a genetic construct comprising aDNA sequence encoding said isolated Family 6 cellulase, which DNAsequence is operably linked to DNA sequences regulating its expressionand secretion from a host microbe, so as to produce recombinant fungalstrains; (ii) selecting the recombinant fungal strains expressing theisolated Family 6 cellulase; and (iii) culturing selected recombinantstrains in submerged liquid fermentations under conditions that inducethe expression of the isolated Family 6 cellulase.